Abstract Junipers are main components of semiarid forests in Central Asia. Conservation of these plant genetic resources should be based on an understanding of factors that have shaped species-level genetic variation. We used Juniperus seravschanica Kom. as a model species to investigate patterns and processes that may be associated with these factors. Novel plastid DNA markers (two minisatellites, one transversion, one indel) were identified and applied to investigate haplotype diversity and population structure in Kyrgyzstan. In total, 540 individuals from 15 populations were analyzed and 11 haplotypes detected. Strong divergence between populations from northern and southern Kyrgyzstan was evident from the haplotype distribution. Gene diversity within populations ranged from 0.083 to 0.765, and was on average higher in southern (0.687) than in northern populations (0.540). A similar pattern was detected in allelic richness. Analysis of molecular variance (AMOVA) revealed that 11.9% of the total genetic variation was due to differences among regions, 1.5% among populations, and 86.6% within populations. NST was not significantly different from GST (0.125), suggesting no evidence of a phylogeographic pattern. A Mantel test detected a weak but significant isolation-by-distance pattern for the whole dataset and southern populations separately. These results suggest that the north of Kyrgyzstan was relatively recently colonized by migrants from southern populations, probably associated with favorable conditions during the early Holocene. The humid Fergana Valley and Fergana Range are probable ecological barriers to gene flow between northern and southern populations.