Allopatric populations living under distinct ecological conditions are excellent systems to infer factors underlying intraspecific venom variation. The venom composition of two populations of Vipera ammodytes, insular with a diet based on ectotherms and mainland with a diet based on ectotherms and endotherms, was compared considering the sex and age of individuals. Ten toxin families, dominated by PLA 2 , svMP, svSP, and DI, were identified through a bottom-up approach. The venom profiles of adult females and males were similar. Results from 58 individual SDS-PAGE profiles and venom pool analysis revealed significant differences between juveniles compared to subadults and adults. Two venom phenotypes were identified: a juvenile svMP-dominated and KUNlacking phenotype and an adult PLA 2 /svMP-balanced and KUN-containing phenotype. Despite differences in prey availability (and, therefore, diet) between populations, no significant differences in venom composition were found. As the populations are geographically isolated, the lack of venom diversification could be explained by insufficient time for natural selection and/or genetic drift to act on the venom composition of island vipers. However, substantial differences in proteomes were observed when compared to venoms from geographically distant populations inhabiting different conditions. These findings highlight the need to consider ecological and evolutionary processes when studying venom variability. Significance: This study provides the first comprehensive analysis of the venom composition of two allopatric populations of Vipera ammodytes, living under similar abiotic (climate) but distinct biotic (prey availability) conditions. The ontogenetic changes in venom composition, coupled with the lack of differences between sex and between populations, shed light on the main determinants of venom evolution in this medically important snake. Seven new proteomes may facilitate future comparative studies of snake venom evolution. This study highlights the importance of considering ecological and evolutionary factors to understand snake venom variation.