Understanding of the genetic relationship within wild lowbush blueberry (Vaccinium angustifolium Ait.) germplasm is important to establish a broad genetic base for safeguarding and for future use of the existing genetic resources. The objective of this study was to assess the genetic variability within 43 wild lowbush blueberry clones, collected from 10 communities of four Canadian provinces, and the cultivar ‘Fundy’ by using inter simple sequence repeat (ISSR) markers, with the hope to establish a reference set of lowbush blueberry germplasm for blueberry conservation, breeding and research. Thirteen primers generated 242 polymorphic ISSR-PCR bands. A substantial degree of genetic similarity was found among the wild collections. Cluster analysis by the unweighted pair-group method with arithmetic averages (UPGMA) separated the 41 genotypes into two main clusters, and identified the three remaining clones as outliers. Furthermore, within one main cluster, the genotypes tended to form sub-clusters that were in agreement with the principal coordinate (PCO) analysis. Geographical distribution contributed to 27% of total variation as revealed by analysis of molecular variance (AMOVA). The ISSR-PCR method was simple, fast and relatively inexpensive to produce useful DNA fragments and detected a sufficient degree of polymorphism to differentiate among lowbush blueberry clones, making this technology valuable for germplasm management and the more efficient choice of parents in current blueberry breeding program.