<i>Objective:</i> To investigate the evolutionary and demographic history of the <i>Gaucho</i>, a distinct population of southern Brazil, relating it to their culture, to assess possible parallel continuity. <i>Methods:</i> Six binary polymorphisms, an Alu insertion polymorphism (YAP) and 12 short tandem repeat loci in the non-recombining region of the Y-chromosome, as well as the sequence of the first hypervariable segment (HVS-I) of the mitochondrial DNA (mtDNA) control region were studied in 150 unrelated males born in the Pampa region of Rio Grande do Sul. <i>Results:</i> Comparison of the results with the other Brazilian and Uruguayan populations, as well as with their putative ancestors, indicated a stronger male Spanish influence than that observed elsewhere in Brazil, a former Portuguese colony. Extensive mtDNA analyses of their Amerindian component gave clear indications of the presence there of material from extinct (Charrua), as well as extant (Guarani) tribes. <i>Conclusions:</i> The genetic analyses contributed in a significant way to reveal that the known cultural continuity between pre- and post-Columbian Pampa populations was also accompanied by an extraordinary genetic continuity.