Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that infects B cells, and its possible presence in humans has garnered increasing attention. This study included 58 participants: 11 with B-cell precursor acute lymphoblastic leukemia (B-ALL, cases) and 47 healthy individuals (controls). Researchers assessed anti-gp51 antibodies and BLV proviral DNA in bone marrow and blood samples. Seropositivity was observed only in the B-ALL group (18.2%; 2/11), while all controls were seronegative. Quantitative PCR targeting the pol gene detected proviral DNA in 74.1% of samples, with similar detection rates between cases and controls. Although proviral load was higher in controls, this difference did not reach statistical significance. Conventional and nested PCR for other viral genes revealed a differential pattern: amplification of the tax gene was significantly associated with B-ALL, whereas gag and env were not. Bayesian Chow–Liu network analyses identified dependencies among viral genes and suggested that contextual factors, such as fieldwork, may influence the association between molecular positivity and B-ALL. Sequence analyses showed that the detected BLV strains clustered with previously reported bovine and human sequences from Colombia, all within genotype 1. These findings support human exposure to BLV and raise important questions about its persistence and potential connections to hematological diseases in humans.