Road-killed mammals, birds, and reptiles were collected weekly from highways in southern Brazil in 2002 and 2005. The objective was to assess variation in estimates of road-kill impacts on species richness produced by different sampling efforts, and to provide information to aid in the experimental design of future sampling. Richness observed in weekly samples was compared with sampling for different periods. In each period, the list of road-killed species was evaluated based on estimates the community structure derived from weekly samplings, and by the presence of the ten species most subject to road mortality, and also of threatened species. Weekly samples were sufficient only for reptiles and mammals, considered separately. Richness estimated from the biweekly samples was equal to that found in the weekly samples, and gave satisfactory results for sampling the most abundant and threatened species. The ten most affected species showed constant road-mortality rates, independent of sampling interval, and also maintained their dominance structure. Birds required greater sampling effort. When the composition of road-killed species varies seasonally, it is necessary to take biweekly samples for a minimum of one year. Weekly or more-frequent sampling for periods longer than two years is necessary to provide a reliable estimate of total species richness.