Studies in nonhuman primates offer information of high relevance to clinical medicine due to their close genetic relationship with humans. Here, we established an optimized protocol for the isolation of antibody V(D)J sequences from rhesus macaque B cells. Nested PCR primers were designed to align to sequences flanking the V(D)J coding region to enable amplification of highly mutated antibody sequences. The primers were evaluated using cDNA from bulk PBMCs as well as from single-sorted memory and naïve B cells from several macaques to ascertain effective germline coverage. The nested PCR efficiency reached 60.6% positive wells for heavy chain amplification, 39.2% for kappa chain, and 23.7% for lambda chain sequences. Matching heavy and light chain sequences, indicating antibodies that potentially can be cloned, were obtained in 50% of the positive wells. Using these primers, we found that the efficiency and specificity of V(D)J amplifications were markedly improved compared to when primers designed for human Ab isolation were used. In particular, the amplification of recombined light chain VJ sequences was improved. Thus, we describe the design and testing of a new set of rhesus-specific primers that enable efficient and specific amplification of heavy, kappa and lambda V(D)J genes from single sorted B cells. The use of these primers will facilitate future efforts to clone and express rhesus macaque MAbs for genetic, functional and structural analyses.