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The genetic variability and population genetic structure of six populations of Praecereus euchlorus and Pilosocereus machrisii were investigated. The genetic variability in single populations of Pilosocereus vilaboensis, Pilosocereus aureispinus, and Facheiroa squamosa was also examined. All of these cacti species have a patchy geographic distribution in which they are restricted to small areas of xeric habitats in eastern Brazil. An analysis of genetic structure was used to gain insights into the historical mechanisms responsible for the patchy distribution of P. euchlorus and P. machrisii. High genetic variability was found at the populational level in all species (P = 58.9 - 92.8%, A(p) = 2.34 - 3.33, H-e = 0.266 - 0.401), and did not support our expectations of low variability based on the small population size. Substantial inbreeding was detected within populations (F-IS = 0.370 - 0.623). In agreement with their insular distribution patterns, P. euchlorus and P. machrisii had a high genetic differentiation (F-ST = 0.484 and F-ST = 0.281, respectively), with no evidence of isolation by distance. Accordingly, estimates of gene flow (N-m) calculated from F-ST and private alleles were below the level of N-m = 1 in P. machrisii and P. euchlorus. These results favored historical fragmentation as the mechanism responsible for the patchy distribution of these two species. The genetic distance between P. machrisii and P. vilaboensis was not compatible with their taxonomic distinction, indicating a possible local speciation event in this genus, or the occurrence of introgression events.