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Background  Studying patterns of introgression can illuminate the role of hybridization in speciation, and help guide decisions relevant to the conservation of rare taxa.  Vipera magnifica  and  Vipera orlovi  are small vipers that have high conservation status due to their rarity and restricted distributions in an area of the Caucasus region where two other related species are present –  V. kaznakovi  and  V. renardi.  Despite numerous observations of hybridization between different species of small vipers, and the potential of a hybrid origin for  V. magnifica  and  V. orlovi  based on their distribution with respect to  V. kaznakovi  and  V. renardi , hypotheses of a hybrid origin have not been formally tested. Here we generate genomic-scale data by performing next generation sequencing of double digest restriction-site associated DNA libraries, and use these multilocus data to test whether these two species are of hybrid origin.    Results  We generated over nine hundred loci for 38 specimens of six taxa, and analysed the dataset using Bayesian clustering and multivariate methods, as well as Patterson D-statistics, which can distinguish between incomplete lineage sorting and introgression as explanations for shared polymorphism. The results demonstrate a pattern of historical admixture in the two purported hybrids that is consistent with past gene flow from  V. renardi  into  V. kaznakovi . The average admixture proportion in individuals was low (6.39 %) in the case of  V. magnifica , but was higher in  V. orlovi  (19.02 %). We also show that the specific individual samples used in D-statistic tests can have a significant impact on inferences regarding the magnitude of introgression, suggesting the importance of including multiple individuals in these analyses.    Conclusions  Our results support the conclusion that both  V. orlovi  and  V. magnifica  had formed through a hybridization event between  V. kaznakovi  and  V. renardi . Given a low proportion of admixture and absence of clear ecological and morphological differences  V. magnifica  should be treated as a marginal population of  V. kaznakovi . Further studies that include analyses of ecological segregation of  V. orlovi  from parental taxa and search for evolutionary consequences of hybridisation would clarify if  V. orlovi  is a distinct hybrid species. Until this we recommend preserving the current taxonomy and protection status of  V. orlovi .    Electronic supplementary material  The online version of this article (doi:10.1186/s12862-016-0647-7) contains supplementary material, which is available to authorized users.

Hybrid origin of European Vipers (Vipera magnifica and Vipera orlovi) from the Caucasus determined using genomic scale DNA markers