A total of 140 Peruvian alpacas of two breeds (Huacaya and Suri) were analyzed for 69 microsatellite markers, in order to make a first approximation to the association analysis with the alpaca fiber diameter, which is the main trait related to alpaca fiber quality. A total of 599 alleles were observed across the two breeds, with a global average of 8.68 alleles per locus. Mean gene diversity in the total population was 0.701, meanwhile both breeds exhibited similar values of 0.686 (Suri) and 0.695 (Huacaya). On the other hand, the values from the inbreeding coefficient (FIS) were 0.154 (Suri) and 0.162 (Huacaya) in both breeds, being the genetic differentiation low between these populations (FST=0.022), and with a gene flow (Nm) value of 9.3. The hierarchical AMOVA corroborated that the differentiation between both breeds only explained the 2.5% of genetic variability.The analysis of association between the microsatellite markers panel and fiber diameter trait was done following an innovative methodology, which was focused in two steps. In the first one, animals were sampled according to a selective genotyping strategy, resulting in six microsatellite markers (LCA68, GLM6, LGU50, VOLP59, LCA85 and LCA90) associated with the genotypes carrying the hypothetical major gene. In the second step, the analysis revealed four significant associations of microsatellite loci (LCA68, VOLP59, LCA9O and GLM6). The subsequent DUNCAN test was used to determine the statistical differences among the fiber diameter EBV means, that corresponded to the different alleles. Eleven out from fourteen alleles of the following loci LCA68 (199, 189, 201, 197, 203 and 205), VOLP59 (112 and110) and LCA90 (243, 229 and 227), showed positive effect (decreasing fiber diameter), and only three alleles for the following loci LCA68 (195) and LCA90 (231 and 249), gave a negative effect (increasing fiber diameter). © 2013 Elsevier B.V.