Seven genetic clades have been recognized within the species Vipera ammodytes (nose-horned viper); however, the precise phylogenetic position of many Balkan populations is unknown. We used Bayesian analysis of the mtDNA sequences from the 16S rRNA mtDNA gene obtained from 47 individuals (26 novel samples sequenced in this study and 21 sequences available from GenBank). Our results show that sampled nose-horned vipers from localities in Serbia are clustered within three clades: the northeastern (23 individuals), the northwestern (two individuals) and the southeastern (one individual). Results revealed an overlapping distribution of the northeastern and the northwestern clades in two populations. We have revealed that the northeastern clade extends further south than previously suggested, to the Ohrid/Prespa lakes in North Macedonia. Our findings contribute to the knowledge of the genetic diversity of this species in Serbia and help to clarify the geographical distributions of mtDNA-defined clades.