Molecular analysis of immunoglobulin variable region (IGV) genes can provide insights into the histogenesis and clonal history of B-cell NHL. We investigated 67 HIV-related NHL (HIV-NHL), including 30 HIV-diffuse large B cell lymphomas (DLBCL), 21 HIV-Burkitt lymphoma (BL), 6 HIV-primary effusion lymphomas (PEL) and 10 HIV-plasmablastic lymphomas (PBL) for usage, mutation frequency and intratumoral heterogeneity of clonal IGV rearrangements as well as mutation profile and CDR3 structure of IGHV, IGKV and IGLV genes. Results were compared to 200 IGV rearrangements from aggressive lymphomas of immunocompetent hosts and to the normal B-cell repertoire. We identified a total of 65 IGHV and 56 IGV light chain rearrangements in HIV-NHL. A functional IGHV rearrangement was found in 60/67 (90%) cases, a functional IGKV chain rearrangement in 17/38 (44.7%) cases and a functional IGLV rearrangement in 21/38 cases (55.3%). Fifty-three of 60 HIV-NHL (88.3%) showed somatic hypermutation in IGHV and/or IGV light chain genes. The average mutation frequency was 9.42% (median 7.50%, range 2.04%–23.3%) for IGHV genes and 5.42% (median 4.20%, range 2.01%–12.5%) for IGV light chain genes. IGV germline rearrangements selectively associated with HIV-PBL (p<0.001). Among mutated cases, average mutation frequencies did not differ among HIV-NHL groups. HIV-NHL showed a significant overrepresentation of the IGHV4 family (28/60; 46.6%) and a significant underrepresentation of IGHV3 family (18/60, 30.0%) compared to aggressive lymphomas of immunocompetent hosts (p<0.05) and to normal B-cells (p<0.05). IGHV4–34 was the IGHV gene most frequently rearranged (17/60; 28.3%) and was overrepresented in HIV-NHL versus aggressive lymphoma of immunocompetent hosts (17%; p<0.03) and normal B-cells (4%; p<0.001). IGHV4-34 expressing cells preferentially associated with lambda chain rearrangements (70%). The IGKV4-1 gene was the IGKV segment most frequently rearranged (6/17; 35.3%) and its usage was biased in HIV-NHL compared to normal B-cells (5.30; p<0.001). The single IGLV gene most frequently encountered was IGLV1-44 (6/17; 35,3%). Distribution of replacement and silent mutations in IGHV sequences showed tendency to conserve FR sequences and maintain antigen binding in 34/52 (65.4%) cases. A higher than expected number of CDR replacement mutations, suggesting selection for high affinity antigen binding, occurred in 17/52 (32.7%) cases. Analysis of intraclonal heterogeneity showed the presence of ongoing mutations in only 1 HIV-BL and 2 HIV-DLBCL. Implications of these data are multifold. First, most HIV-NHL derive from B-cells persistently subjected to GC reaction, suggesting a potential role for antigen stimulation in the pathogenesis of these lymphomas. This hypothesis is supported by the finding of antigen binding preservation in the majority of HIV-NHL and selection for high affinity antigen binding in a fraction of cases. Second, the preferential usage of IGHV4-34 and IGKV4-1 genes in a fraction of HIV-NHL may suggest a role for stimulation of pre-neoplastic B-cells with polyreactive and/or autoreactive antigens. Finally, at variance with NHL of immucompetent hosts, the presence of intraclonal heterogeneity is a rare finding in HIV-NHL, suggesting a derivation from B-cells that have concluded the GC-reaction.