Simple SummaryThis research employs fourteen and twelve microsatellite markers for two Huacaya alpaca populations from Southern Peru to estimate the genetic diversity and population structure. Our findings show that these two populations (Quimsachata and Ajoyani) were not structured. We also found that both populations have a large amount of diversity, which is a good indicator for conservation measures. These findings provide useful indicators regarding conservation programs created for this species to ensure their sustainable use.AbstractThe alpaca population mostly consists of the Huacaya phenotype and is widely distributed in Southern Peru. This study aimed to estimate the genetic diversity and population structure of two Huacaya alpaca populations (Ajoyani and Quimsachata) using fourteen and twelve microsatellite markers for each population, respectively. A total of 168 alpaca biological samples were outsourced to Peruvian laboratories for DNA extraction and genotyping. For genetic diversity, observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC), and fixation indices values were estimated. An admixture analysis was performed for the population structure analysis. Different programs were used for these estimations. In total, 133 (Ajoyani) and 129 (Quimsachata) alleles were found, with a range of 4 to 17 by locus. The mean HO, HE, and PIC per marker for Ajoyani were 0.764 ± 0.112, 0.771 ± 0.1, and 0.736; for Quimsachata, they were 0.783 ± 0.087, 0.773 ± 0.095, and 0.738, respectively. The population structure showed no structure with K = 2. This study provides useful indicators for the creation of appropriate alpaca conservation programs.