Hemoglobin profile studies have been carried out in four samples from different districts of Porto Velho (Rondônia State) in the western Amazonian region of Brazil: Candelária, Bate Estaca, Hemeron (at the State Blood Bank), and São Carlos. Samples from 337 unrelated individuals were collected during medical and paramedical team visits by professionals from the Instituto de Pesquisa em Patologia Tropical and the Centro de Pesquisa em Patologias Tropicais (both research institutes in tropical diseases). The aim of this study is to assess the frequency of alleles in the hemoglobin system, mainly alleles HB*A, *S, and *E. The overall phenotype frequencies were HB A,S = 0.025, HB A,E = 0.006, and HB A,A = 0.969. Samples from the blood bank subjects and samples from the homogeneous areas of São Carlos and Candelária plus Bate Estaca have a chi-square of heterogeneity of 6.383 (p = 0.041) and 8.406 (p = 0.015), respectively. The allele frequencies (HB*A = 0.984, HB*S = 0.012, and HB*E = 0.003) do not significantly differ from frequencies found in other Brazilian regions.