We analyzed the genotypes of Eurasian lynx (Lynx lynx) from three populations in the westernmost part of the species main range. One population was situated at the distribution edge (NE Poland) and the two other (Latvia and Estonia) were located within the main, contiguous range of the species. The aim was to determine if the genetic composition varied among these populations and if there was evidence of isolation among them. Based on microsatellite allele frequencies, we found the allelic richness in Polish lynx to be lower than that in lynx from Latvia and Estonia. We also found significant differentiation among the lynx populations, with the NE Poland population forming a distinct genetic group relative to the two other populations (R (ST) = 0.15 and 0.22, P < 0.0001). We suggest that genetic differentiation among lynx populations is the result of habitat insularisation that limits gene flow. This finding emphasizes the necessity to consider the lynx genetic differentiation in conservation planning of this species in Poland.