OBJECTIVE To determine titers of serum antibodies against 3 genotypes of bovine parainfluenza 3 virus (BPI3V) in unvaccinated ungulates in Alabama. ANIMALS 62 cattle, goats, and New World camelids from 5 distinct herds and 21 captured white-tailed deer. PROCEDURES Serum samples were obtained from all animals for determination of anti-BPI3V antibody titers, which were measured by virus neutralization assays that used indicator (reference) viruses from each of the 3 BPI3V genotypes (BPI3V-A, BPI3V-B, and BPI3V-C). The reference strains were recent clinical isolates from US cattle. Each sample was assayed in triplicate for each genotype. Animals with a mean antibody titer = 2 for a particular genotype were considered seronegative for that genotype. RESULTS Animals seropositive for antibodies against BPI3V were identified in 2 of 3 groups of cattle and the group of New World camelids. The geometric mean antibody titer against BPI3V-B was significantly greater than that for BPI3V-A and BPI3V-C in all 3 groups. All goats, captive white-tailed deer, and cattle in the third cattle group were seronegative for all 3 genotypes of the virus. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that BPI3V-A may no longer be the predominant genotype circulating among ungulates in Alabama. This may be clinically relevant because BPI3V is frequently involved in the pathogenesis of bovine respiratory disease complex, current vaccines contain antigens against BPI3V-A only, and the extent of cross-protection among antibodies against the various BPI3V genotypes is unknown.