The Cactaceae comprises approximately 1800 species, found mostly in the tropical deserts of North and South America. The family has been the object of intensive study but it is not until recently that molecular tools have been used to elucidate its phylogenetic relationships. Most of the Cactaceae found in the Central Andes of Peru and Bolivia belong to tribe Trichocereeae. Phylogenetic relationships within the tribe have been historically controversial, therefore, a molecular study using chloroplast and nuclear markers was designed to elucidate phylogenetic relationships among species of Haageocereus and the relationship of this genus to the rest of taxa in the Trichocereeae. We sequenced cpDNA (rpoB, rpl16, and 23S) and nrDNA (ITS1 and ITS2) of 107 ingroup taxa comprising the Trichocereeae and 11 outgroup taxa from different tribes. The analyses support a monophyletic Trichocereeae (including Praecereus euchlorus), the exclusion of seven genera found to be more closely related to Brazilian Cereeae, and the maintenance of several genera historically placed in Trichocereeae, including Haageocereus and Espostoa. Based on our results, the taxonomy of the Trichocereeae does not reflect its phylogeny and is in need of revision. We describe 19 polymorphic dinucleotide microsatellite loci isolated from two species of Haageocereus ( H. tenuis and H. pseudomelanostele) that have probed their utility in the analysis of population differentiation and genetic diversity throughout Haageocereus, as well as in the analysis of clonal and hybrid individuals. Chromosome numbers for a total of 54 individuals representing 14 genera and 39 species of Cactaceae, mostly in tribe Trichocereeae, are reported, demonstrating the presence of polyploidy in the Trichocereeae and its dominance in certain genera. Clonal propagation and apomixis in the triploid Haageocereus tenuis has significant implications for the evolutionary biology and ecology of Haageocereus and other clonal Cactaceae.