Secondary sexual displays may be overlooked in many species, especially when they are ephemerally expressed or imperceptible to human senses. Zebrafish (Danio rerio), like many schooling fish, do not appear sexually dichromatic, but previous anecdotal observations indicate that sexual colouration is expressed briefly during courtship (ephemeral nuptial colouration). Our goals were to compare colour estimates of male vs. female zebrafish using digital photography in situ, computer software and human observations. We found that both sexes changed their colour estimates during spawning (dark and light stripes) and that some sex differences (light stripes) were larger or only became apparent during this time. We also found that individual males that appeared more colourful and conspicuous to the human eye engaged in courtship more often than less conspicuous males. We detected differences in the colour estimates between wild-derived vs. a laboratory strain of zebrafish and reduced individual variation in the laboratory strain. This is the first study to systematically and objectively quantify body colour in zebrafish by utilizing colour estimates, although further studies are needed to determine the underlying mechanisms and signalling functions of this sexual dichromatism.