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Tepary bean (Phaseolus acutifolius A. Gray) is a dry-land crop species that originated in the deserts of Mexico and the south-western United States and therefore is proposed as a source of drought and salt tolerance for related species and for production in marginal rainfall areas. Few genetic tools have been developed or tested for tepary bean but microsatellites from common bean are an obvious choice for diversity analysis in the crop. The first goal of this study was to validate a set of gene-derived and non-gene simple sequence repeat or microsatellite markers from common bean in tepary bean cultivars and wild relative accessions. The second and more extensive objective of this study was to evaluate the genetic diversity and population structure of the tepary bean accessions to determine if leaf-morphology variants are valid as separate sub-groups of wild tepary beans; if P. parvifolius exist as a separate variants or species; and if cultivated tepary beans originated from one domestication event or several events. Our analysis of 140 tepary bean genotypes showed that a single domestication was likely as the cultivars were most closely related to accessions from Sinaloa and northern Mexico and that diversity was much higher in the wild genotypes compared to the cultivated ones. Other results were that P. parvifolius was classified as a separate species by population structure analysis while the variants P. acutifolius var. acutifolius and var. tenuifolius were admixed and inter-crossed. P. latifolius is not a valid species or variant of P. acutifolius but represents a group of cultivars within tepary bean. This is the first analysis of microsatellite diversity in tepary beans and has implications for breeding and conservation of this crop and its wild relatives.