• Premise of the study: Microsatellites were isolated from two species of the genus Haageocereus (H. tenuis and H. pseudomelanostele) to be applied in studies of genetic diversity and population structure. • Methods and Results: Five loci were employed in a preliminary study of genetic diversity and population differentiation in two rare (H. tenuis and H. repens) and two widespread (H. acranthus and H. pseudomelanostele) species, yielding between one and 44 alleles per locus. All five loci were polymorphic, with overall levels of observed and expected heterozygosities ranging from 0.478 to 0.871 and from 0.564 to 0.956, respectively. Three additional loci were scored in H. pseudomelanostele. These eight plus the remaining 11 loci were amplified from putative parents of three hybrids involving Haageocereus and Espostoa. • Conclusions: These markers will facilitate analysis of genetic diversity, hybridization, and population differentiation throughout Haageocereus and Espostoa. © 2010 Botanical Society of America.