Bufo spinulosus has a wide and fragmented distribution range in Chile (18degrees to 33degrees S) along altitudinal and latitudinal gradients. Genetic variation was estimated using RAPD (Random Amplified Polymorphic DNA) markers in 10 populations from northern and central Chile. Morphometric and genetic information was analyzed as a function of geographical origin. The correlation between genetic and morphometric differentiation was analyzed by the Mantel test. An increase in body size as a function of latitude was observed. Specimens from El Tatio had the smallest body size and the greatest morphometric divergence. The AMOVA applied to genetic data indicated that 57.85 % of the variance is explained by interregional differences and that 30.12 % of the variance is found within populations. Low levels of within-regions genetic differentiation was observed in northern populations while higher levels of genetic differentiation was found in populations from central Chile. Mantel tests revealed a significant, positive correlation between genetic variation and geographic distance. When we excluded El Tatio population, Mantel test analyses showed significant correlations between morphological distance and genetic and geographic distances. We discuss whether water temperature could explain the morphological divergence observed in individuals from El Tatio.