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Restriction fragment length polymorphisms are good anthropological markers for discriminating geographically distinct populations at both the allele and the haplotype level. Two communities of African ancestry and ladinos, mestizos, and mulattoes living in the Esmeraldas province in northwestern Ecuador were analyzed for three RFLPs (EcoRI, RsaI, and MspI) of the COL1A2 gene. Also, the same markers were studied in a population sample from Spain to compare the allele and haplotype frequencies of the Esmeraldas populations with those of their representative European parental population. Data for the native American and sub-Saharan African founder components were available from the literature. No significant levels of differentiation between the two African Ecuadoran communities emerged from either the frequency analysis of each single marker and all three RFLP markers together or from the AMOVA. The ladinos and mestizos also showed a rather similar distribution of allele and haplotype frequencies, confirming that the two ethnic terms do not correspond to genetically different populations. The comparison with the supposed founding European, sub- Saharan African, and native American populations indicated a large presence of African genes in the gene pool of both communities, with a higher proportion of the Amerindian component in Viche than in Rio Cayapas. The present findings confirm the previous genetic admixture estimates based on nuclear and mitochondrial DNA markers and the demographic data.