The demand for B-virus-free animals for biomedical research is increasing, while at the same time the availability of such animals is decreasing. The establishment of Specific Pathogen-Free (SPF) breeding macaque colonies is a priority of the National Institutes of Health. Nevertheless, it is well known that seroreactivity to B-virus can be difficult to interpret, particularly as it can vary over time in a single animal. The aim of the present study was to implement a reliable algorithm to examine B-virus reactivity among the rhesus monkey population of the Caribbean Primate Research Center. The sensitivity and specificity of our assay were determined using reports from two different laboratories as references. Whereas 95.4% of animals showed consistent serological status and 4.6% of animals recruited to this SPF program showed serovariability to B-virus over the initial 2 years of examination. Implications for all SPF programs are discussed in this article.