Road mortality is probably the best-known and visible impact of roads upon wildlife. Although several factors influence road-kill counts, carcass persistence time is considered the most important determinant underlying underestimates of road mortality. The present study aims to describe and model carcass persistence variability on the road for different taxonomic groups under different environmental conditions throughout the year; and also to assess the effect of sampling frequency on the relative variation in road-kill estimates registered within a survey. Daily surveys of road-killed vertebrates were conducted over one year along four road sections with different traffic volumes. Survival analysis was then used to i) describe carcass persistence timings for overall and for specific animal groups; ii) assess optimal sampling designs according to research objectives; and iii) model the influence of road, animal and weather factors on carcass persistence probabilities. Most animal carcasses persisted on the road for the first day only, with some groups disappearing at very high rates. The advisable periodicity of road monitoring that minimizes bias in road mortality estimates is daily monitoring for bats (in the morning) and lizards (in the afternoon), daily monitoring for toads, small birds, small mammals, snakes, salamanders, and lagomorphs; 1 day-interval (alternate days) for large birds, birds of prey, hedgehogs, and freshwater turtles; and 2 day-interval for carnivores. Multiple factors influenced the persistence probabilities of vertebrate carcasses on the road. Overall, the persistence was much lower for small animals, on roads with lower traffic volumes, for carcasses located on road lanes, and during humid conditions and high temperatures during the wet season and dry seasons, respectively. The guidance given here on monitoring frequencies is particularly relevant to provide conservation and transportation agencies with accurate numbers of road-kills, realistic mitigation measures, and detailed designs for road monitoring programs.