BK polyomavirus (BKV) causes polyomavirus-associated nephropathy (PyVAN) and polyomavirus-associated hemorrhagic cystitis (PyVHC) following kidney transplantation and allogeneic hematopoietic stem cell transplantation (HST). BKV strains fall into four distinct genotypes (BKV-I, -II, -III, and -IV) with more than 80% of individuals are seropositive against BKV-I genotype, while the seroprevalence of the other four genotypes is lower. PyVAN and PyVHC occurs in immunosuppressed (e.g. transplant recipients) or immunomodulated (e.g. pregnant women) individuals. In the case of transplant patients, the sole treatment is to reduce immunosuppression, which increases the chance of graft failure. Multiple investigations have shown that polyclonal antibodies have a role in preventing or treating BKV-mediated sickness, implying that a broadly reactive monoclonal antibody (mAb) regimen targeting BKV could be used to limit virus propagation in varied patient populations. Thus, we utilized a heterologous immunization strategy using BKV genotype I-IV major capsid protein VP1 DNA in transgenic VelocImmune@ mice to generate broadly reactive anti-BKV antibodies. Hybridoma clones from the immunized mice were screened using high-throughput binding assays against VP1 of the various BK genotypes. The binding clones were then assessed for neutralization of BKV pseudoviruses consisting of the VP1 protein of the BKV-I, -II, -III, or -IV genotypes. Overall, the screening identified more than 170 genotype-specific mAbs, as well as 15 broadly cross-neutralizing mAbs against BKV-I, -II, -III, and - IV PsVs. The unique panel of broadly neutralizing mAbs could be utilized prophylactically or therapeutically to prevent or treat BKV-induced illnesses.