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A common classification of the Lepidoptera involves their differentiation into butterflies and moths. Butterflies are a natural monophyletic group, often given the suborder Rhopalocera, which includes Papilionoidea (true butterflies), Hesperiidae (skippers), and Hedylidae (butterfly moths). In this taxonomic scheme, moths belong to the suborder Heterocera. Other taxonomic schemes have been proposed, the most common putting the butterflies into the suborder Ditrysia and then the "superfamily" Papilionoidea and ignoring a classification for moths.

The most obvious difference is in the feelers, or antennae. Most butterflies have thin slender filamentous antennae which are club shaped at the end. Moths, on the other hand, often have comb like or feathery antennae, or filamentous and unclubbed. This distinction is the basis for the earliest taxonomic divisions in the Lepidoptera: the Rhopalocera ("clubbed horn", the butterflies) and the Heterocera ("varied horn", the moths). There are, however, exceptions to this rule and a few moths (the families Castniidae, Uraniidae, Apoprogonidae, and Sematuridae) have clubbed antennae. Some butterflies, like Pseudopontia paradoxa from the forests of central Africa, lack the club ends. The hesperiids often have an angle to the tip of the antenna.

Many moths have a frenulum which is a filament arising from the hindwing and coupling (matching up) with barbs on the forewing. The frenulum can be observed only when a specimen is in hand. There is only one known species of butterfly with a frenulum, which is the male regent skipper Euschemon rafflesia. Some moths have a lobe on the forewing called a jugum that helps in coupling with the hindwing. Butterflies lack these structures.

Most moth caterpillars spin a cocoon made of silk within which they metamorphose into the pupal stage. Most butterfly caterpillars, on the other hand, form an exposed pupa made from a hardened protein, also termed a chrysalis. There are many exceptions to this rule, however. For example, the hawk moths form an exposed pupa which is underground. Spongy moths sometimes form butterfly style pupae, hanging on twigs or tree bark, although usually they create flimsy cocoons out of silk webbing and leaf bits, leaving the pupa exposed. The plume winged moths of the family Pterophoridae also pupates without a cocoon and the pupa resembles the chrysalis of the pierid butterfly. A few skipper butterfly larvae also make crude cocoons in which they pupate, exposing the pupa a bit. The Parnassius butterfly larvae make a flimsy cocoon for pupation and they pupate near the ground surface between debris.

Most butterflies have bright colours on their wings. Nocturnal moths on the other hand are usually plain brown, grey, white or black and often with obscuring patterns of zigzags or swirls which help camouflage them from predators as they rest during the day. However, many day flying moths are brightly coloured, particularly if they are toxic. These diurnal species evolved to locate their mates visually and not primarily by pheromone as their drab nocturnal cousins. There are several exceptions to this rule, such as with the diurnal Zygaenidae and Sytomidae families of moths, both of which have apposition eyes, or the Hedyloidea family of butterflies, which are nocturnal and feature superposition eyes. In most cases where one species is found to be using the opposite type of eye than expected, it is because they are active during the opposite time of day than is normal for other butterflies or moths.

Most moths are nocturnal or crepuscular while most butterflies are diurnal. There are however exceptions, including the spectacular Uraniidae or sunset moths. A few species, such as the male European/North American spongy moth, fly during both day and night in search of the females, which are flightless.

Moths usually rest with their wings spread out to their sides. Butterflies frequently fold their wings above their backs when they are perched although they will occasionally "bask" with their wings spread for short periods (several types of Swallowtail butterflies tend to frequently rest with their wings spread when in sunlight). However, some butterflies, like the skippers, may hold their wings either flat, or folded, or even in between (the so called "jet plane" position) when perched. Most moths also occasionally fold their wings above their backs when they are in a certain spot (like when there is no room to fully spread their wings). A sometimes confusing family can be the Geometridae (such as the winter moth) because the adults often rest with their wings folded vertically. These moths have thin bodies and large wings like many butterflies but may be distinguished easily by structural differences in their antennae (e. g. bipectinate).