Selective breeding of the guanaco and vicuña indisputably led to the domestic llama and alpaca. However, the place, time and process of domestication remain poorly documented, exacerbated by increased evidence of historic and recent introgression among both domestic populations, especially those involving alpaca. Here, we report the first comprehensive assessment of patterns of microsatellite variation of 13 polymorphic microsatellites in 797 individuals representing 4 species and 2 subspecies to estimate historic and ongoing patterns of gene flow. Our results confirm that llamas were domesticated almost exclusively from the northern subspecies of guanaco (Lama guanicoe cacsilensis), but cluster analyses did not support previous findings that alpacas are solely derived from the northern subspecies of vicuña (Vicugna mensalis), with some more-recent admixture with southern populations. We also confirmed continued gene flow between llama and alpaca, and found evidence for crosses between presumably feral llamas and guanacos in northern Chile. Our results highlight the need to develop and test more-accurate markers and tools for assessing the genetic heritage of individuals, to link phenotypic variation with genetic ancestry, and to conserve historic patterns of unique variation among both the wild and domestic populations.