There is a need for classifying and conserving local apple cultivars from two main regions in Bosnia and Herzegovina (B&H). Consequently, 71 local apple accessions (31 from Sarajevo and 40 from eastern Bosnia) were evaluated with a set of 10 simple sequence repeats (SSRs). These accessions were compared with 37 reference cultivars (24 traditional B&H and 13 international cultivars maintained at the ex situ collection Srebrenik) to determine synonyms, homonyms, and possible introgression of foreign genotypes into the local apple germplasm. Using 10 primer pairs of microsatellites, we were able to amplify 135 alleles for the 71 local apple accessions. Detection of more than two different alleles per locus was observed for 34 accessions. Fourteen different homonyms and 12 synonyms were identified among all the apple cultivars analyzed. Analysis of molecular variance (AMOVA) revealed a significant genetic differentiation between most of the groups analyzed but not between accessions from Sarajevo and eastern Bosnia. Bayesian method and admixture analysis of the allele frequency allowed classification of all accessions analyzed and found that they fell into two main groups [reconstructed panmictic populations (RPPs)]. Strong genetic differentiation between these two groups was detected using AMOVA (fCT = 0.130; P < 0.001). Analysis of the genetic structure indicates that overall, approximately half of the local apple cultivars from Sarajevo and eastern Bosnia (52% and 45%, respectively) grouped in the RPP1 consisting mainly out of international reference cultivars, whereas the other half grouped in the RPP2 with traditional B&H reference cultivars. Both neighbor joining (NJ) cluster analysis based on Bruvo genetic distance and factorial correspondence analysis (FCA) confirmed the results of the genetic structure analysis. The molecular data show that both apple accessions from Sarajevo and from eastern Bosnia represent an interesting source of diversity, which needs to be conserved.