Retroviral DNA integration shows distinct target site preferences among avian sarcoma-leukosis virus (ASLV), human immunodeficiency virus (HIV), and murine leukemia virus (MLV). The study analyzed 3,127 integration site sequences from human cells and compared the integration preferences of these three retroviruses. HIV vectors strongly favored active genes in both primary cells and cell lines, while MLV showed a strong bias for integration near transcription start sites. ASLV showed no significant preference for active genes or transcription start regions. The study also found that integration site selection was influenced by tissue-specific transcription, with HIV integration showing tissue-specific targeting. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were interleaved with unfavorable regions at CpG islands. The results suggest that virus-specific binding of integration complexes to chromatin features likely guides site selection. The study highlights the importance of understanding retroviral integration preferences for safe gene therapy and the potential for different retroviral vectors to be used in different therapeutic contexts.Retroviral DNA integration shows distinct target site preferences among avian sarcoma-leukosis virus (ASLV), human immunodeficiency virus (HIV), and murine leukemia virus (MLV). The study analyzed 3,127 integration site sequences from human cells and compared the integration preferences of these three retroviruses. HIV vectors strongly favored active genes in both primary cells and cell lines, while MLV showed a strong bias for integration near transcription start sites. ASLV showed no significant preference for active genes or transcription start regions. The study also found that integration site selection was influenced by tissue-specific transcription, with HIV integration showing tissue-specific targeting. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were interleaved with unfavorable regions at CpG islands. The results suggest that virus-specific binding of integration complexes to chromatin features likely guides site selection. The study highlights the importance of understanding retroviral integration preferences for safe gene therapy and the potential for different retroviral vectors to be used in different therapeutic contexts.