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Eritrea is a multi-ethnic country of over 3 million of people consisting of different ethnic groups, having each its own language and cultural tradition. Due to the lack of population genetic data for markers of forensic interest, in this study, we analyzed the genetic polymorphisms of 23 Y-chromosome STR loci and of 12 X-chromosome STR loci in a sample of 255 unrelated individuals from 8 Eritrean ethnic groups, with the aim to generate a reference haplotype database for anthropological and forensic applications. X- and Y-chromosomes markers may indeed offer information especially in personal identification and kinship testing, when relying on the availability of large local population data to derive sufficiently accurate frequency estimates. The population genetic analyses in the Eritrean sample for both the two set of Y- and X-STR markers showed high power of discrimination both at country-based and population levels. Comparison population results highlight the importance of considering the ethnic composition within the analyzed country and the necessity of increasing available data especially when referring to heterogeneous populations such as the African ones.