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The genetic structure of the population of Alia (Sicily, Italy) was analyzed using 15 short tandem repeats: TPOX, D2S1338, D3S1358, FIBRA, D5S818, CSF1PO, D7S820, D8S1179, TH01, VWA, D13S317, D16S539, D18S51, D19S433, and D21S11. Two of these markers, D2S1338 and D19S433, have never before been used in research on population genetics and only recently have they been put to use in forensic medicine. Results of the analysis underline the genetic isolation of the Alia population and show it to be a recent bottleneck as a consequence of a cholera epidemic in 1837. While comparing the Alia population with other populations from Sicily, a genetic heterogeneity within Sicily was uncovered, thus confirming previous results obtained from the analysis of classical markers. This heterogeneity underlines the existence of genetic boundaries within the island. Comparisons with other Italian, Mediterranean, and European populations highlight the differentiation of the Sicilian population, reflecting the presence of a genetic boundary that separates Sicily from northern and central Italy and from the western Mediterranean basin.