The effects of long-term habitat fragmentations on genetic and population differentiation of Betula pendula Roth were investigated using chloroplast DNA (cpDNA) variations. Leaf samples were collected from four small remnant populations across the north of Iran. Three pairs of universal primers were used to amplify cpDNA, large single copy regions of trnC-trnD, trnK1-trnK2 and trnD-trnT. A total of 18 of the cpDNA haplotypes in the four populations were identified, however, no clear phylogeographic structuring of haplotypes could be detected. The total genetic diversity (HT) for all populations was high (0.932). Average intra-population genetic diversity was estimated as HS = 0.729 and average differentiation of populations GST = 0.218. Mantel tests of isolation by distance revealed a significant relationship between Wright’s inbreeding coefficient (Fst) and geographical distances for the four populations in Iran (r = 0.77, p < 0.05). The results of the hierarchical analysis of molecular variance (AMOVA) indicated that a 66% variation was partitioned within populations, whilst the variance amongst the four populations was only 34%. We suggest that significant genetic differentiation amongst populations can likely be attributed to reduced gene flow as a result of habitat fragmentation.