The early events of the HIV replication cycle involve the interaction between viral envelope glycoproteins and their cellular CD4-chemokine (CCR5/CXCR4) receptor complex. In this study, for the first time, the HIV-2 A-subtype gp125(C2-V3-C3) mutations and their tropism association were characterized by analyzing 149 HIV-2 sequences from the Los Alamos database. The analysis has strengthened the importance of C2-V3-C3 region as a determinant factor for co-receptor selection. Moreover, statistically significant correlations were observed between C2-V3-C3 mutations, and several correlated mutations were associated with CXCR4 and CCR5 co-receptor usage. A dendrogram showed two distinct clusters, with numerous associated mutations grouped, thus dividing CCR5- and CXCR4-tropic viruses. Fourteen X4-tropic virus mutations, all in V3 and C3 domains and forming highly significant subclusters, were found. Finally, R5 associations, two strong subclusters were observed, grouping several C2-V3-C3 mutated positions. These data indicate the possible contribution of C2-V3-C3 mutational patterns in regulating HIV-2 tropism.