In this work, we present new evidence supporting the idea that the first Americans were very distinct from late and recent Native Americans and Asians in terms of cranial morphology. The study is based on 30 early Holocene specimens recovered from Sumidouro Cave (Lagoa Santa region, central Brazil) by Peter Lund in 1843. Sumidouro is the largest known collection of Paleoindian skulls deriving from a single site. Six different multivariate statistical methods were applied to assess the morphological affinities of the Sumidouro skulls in comparison to Howells' worldwide extant series and late archaic Brazilian series (Base Aérea and Tapera). The results show a clear association between Sumidouro and Australo-Melanesians and none with late Asian and Amerindian series. These results are in accordance with those of previous studies of final Pleistocene/early Holocene human skulls from South, Central, and North America, attesting to a colonization of the New World by at least two different, succeeding biological populations: an early one with a cranial morphology similar to that found today in the African and Australian continents, and a later one with a morphology similar to that found today among northeastern Asians.