At least 25 African-derived populations (quilombo remnants) are believed to exist in the Ribeira River Valley, located in the southern part of São Paulo State, Brazil. We studied four Alu polymorphic loci (APO, ACE, TPA25, and FXIIIB) in individuals belonging to six quilombo remnants in addition to individuals sampled from the city of São Paulo. The allelic frequencies observed in the quilombo remnants were similar to those previously observed in African-derived populations from Central and North America. Genetic variability indexes (Fst and Gst values) in our quilombos were higher than the reported values for the majority of other populations analyzed for the same kind of markers, but lower than the variability usually observed in Amerindian groups. The observed high degree of genetic differentiation may be due to genetic drift, especially the founder effect. Our results suggest that these populations behave genetically as semi-isolates. The degree of genetic variability within populations was larger than among them, a finding described in other studies. In the neighbor-joining tree, some of the Brazilian quilombos clustered with the African and African-derived populations (São Pedro and Galvão), others with the Europeans (Pilões, Maria Rosa, and Abobral). Pedro Cubas was placed in an isolated branch. Principal component analysis was also performed and confirmed the trends observed in the neighbor-joining tree. Overall, the quilombos showed a higher degree of gene flow than average when compared to other worldwide populations, but similar to other African-derived populations. Am. J. Hum. Biol. 16:264–277, 2004. © 2004 Wiley-Liss, Inc.