Mouse mammary tumor virus (MMTV) is a betaretrovirus that plays a causal role in the development of breast cancer and lymphoma in mice. Closely related sequences that share 91–99% nucleotide identity with MMTV have been repeatedly found in humans with neoplastic and inflammatory diseases. Evidence for infection with a betaretrovirus has been found in patients with breast cancer and primary biliary cholangitis and referred to as the human mammary tumor virus and the human betaretrovirus (HBRV), respectively. Using the gold standard technique of demonstrating retroviral infection, HBRV proviral integrations have been detected in cholangiocytes, lymph nodes, and liver of patients with primary biliary cholangitis. However, the scientific biomedical community has not embraced the hypothesis that MMTV like betaretroviruses may infect humans because reports of viral detection have been inconsistent and robust diagnostic assays are lacking. Specifically, prior serological assays using MMTV proteins have produced divergent results in human disease. Accordingly, a partial HBRV surface (Su) construct was transfected into HEK293 to create an ELISA. The secreted HBRV gp52 Su protein was then used to screen for serological responses in patients with breast cancer and liver disease. A greater proportion of breast cancer patients (n = 98) were found to have serological reactivity to HBRV Su as compared to age- and sex-matched control subjects (10.2% versus 2.0%, P=0.017, OR = 5.6 [1.25–26.3]). Similarly, the frequency of HBRV Su reactivity was higher in patients with primary biliary cholangitis (n = 156) as compared to blood donors (11.5% vs. 3.1%, P=0.0024, OR = 4.09 [1.66–10.1]). While the sensitivity of the HBRV Su ELISA was limited, the assay was highly specific for serologic detection in patients with breast cancer or primary biliary cholangitis, respectively (98.0% [93.1%–99.7%] and 97.0% [93.4%–98.6%]). Additional assays will be required to link immune response to betaretrovirus infection and either breast cancer or primary biliary cholangitis.