Associations between the evolutionary loss or gain of colours and habitat can be informative about how environment drives colour evolution, and provide insight into their functions. Despite a wealth of studies on colours, the function of many colour patches are not well understood. This is especially the case for dark facial markings. Dark facial stripes are common in snakes, including many pitvipers, which possess facial stripes that extend from the eye towards the corner of the mouth. We investigated whether the evolutionary loss or gain of facial stripes in New World pitvipers is associated with activity period and habitat, hoping to shed light on their function. First, we examined whether evolutionary loss or gain of facial stripes is associated with nocturnal or diurnal activity. It has been suggested that facial stripes may protect the venom from ultraviolet radiation, which would be higher in diurnal species. Similarly, snakes in open habitats may experience higher ultraviolet radiation than those in closed habitats. Alternatively, we examined whether evolutionary loss or gain of facial stripes is associated with terrestrial or non-terrestrial habitat use. Studies on similar facial markings in other vertebrates indicated that they have a signalling function. If facial stripes in pitvipers have a signalling function, arboreal or saxicolous habitat use may limit signal effectiveness. Hence, we hypothesized that there may be an association between facial stripe evolution and habitat use. Using combined phylogenies that included almost every New World pitviper species, we tested correlations between facial stripes and ultraviolet exposure (activity period and habitat openness) and habitat use (terrestrial or non-terrestrial) using concentrated-changes tests and Pagel's correlation tests. Our data did not support the hypothesis that ultraviolet exposure influenced facial stripe evolution. Instead, the evolutionary loss of facial stripes was associated with non-terrestrial habitat use, such as arboreal and saxicolous habitats. In these habitats, we suggest the effectiveness of facial stripes as a signal would be limited, leading to their evolutionary loss. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104, 923–933.