The region known as pX in the 3′ end of the human T-cell lymphotropic virus type 1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. HTLV-1 ORF-I produces the protein p12 and its cleavage product p8. The functions of these proteins have been linked to immune evasion and viral infectivity and persistence. It is known that the HTLV-1 infection does not necessarily imply the development of pathological processes and here we evaluated whether natural mutations in HTLV-1 ORF-I can influence the proviral load and clinical manifestation of HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). For that, we performed molecular characterization, datamining and phylogenetic analysis with HTLV-1 ORF-I sequences from 156 patients with negative or positive diagnosis for HAM/TSP. Our analyses demonstrated that some mutations may be associated with the outcome of HAM/TSP (C39R, L40F, P45L, S69G and R88K) or with proviral load (P34L and F61L). We further examined the presence of mutations in motifs of HBZ and observed that P45L mutation is located within the HBZ nuclear localization signal and was found more frequently between patients with HAM/TSP and high proviral load. These results indicate that some natural mutations are located in functional domains of ORF-I and suggests a potential association between these mutations and the proviral loads and development of HAM/TSP. Therefore it is necessary to conduct functional studies aimed at evaluating the impact of these mutations on the virus persistence and immune evasion.