HPV-16 is the most prevalent human papillomavirus genotype found in cervical intraepithelial neoplasias. The regulatory region of the HPV genome, LCR, has several binding sites for cellular and viral transcription factors, and nucleotide substitutions in this genomic region can interfere with the viral oncogenic expression. The present study aims to determine the LCR variability of European and non-European HPV-16 variants found in Brazil. Through automated sequencing, it was possible to characterize the LCR of ten non-European (eight Asian-American, one African 1, one African 2) and twelve European isolates. Among the 22 isolates analyzed, nine may be new variants of HPV-16, with different combinations of previously reported nucleotide substitutions, and three showed new substitutions not previously reported. Two new nucleotide substitutions, the insertion of T at position 7621 and the substitution of A to G at position 7836, were found in a single isolate, Bsb-14, a putative new African 1 variant. The characterization of the LCR of human papillomaviruses can be of pivotal importance to the understanding of the viral replication and pathogenicity.