CD4 T follicular helper (Tfh) cells play a unique and essential role in the generation of B cell responses in the lymph node microenvironment. Here we sought to determine if differential expression of PD-1 could be used to delineate Tfh cells in rhesus macaque lymph nodes (LN). CD3(+)CD4(+) T cells were found to harbor a unique subset of cells that expressed the Program death-1 (PD-1) receptor at significantly high levels that were enriched in the LN compartment as compared to peripheral blood. The LN CD4(+)PD1(hi) T cells expressed a predominantly CD28(+)CD95(+) central memory phenotype and were CCR7(lo)ICOS(hi)Bcl6(hi). Additionally, CD4(+)PD1(hi) T cells preferentially expressed high levels of CXCR5 and IL-21 and significantly correlated with Bcl6(+)Ki-67(+) IgG(+) B cells. As Bcl6 is primarily expressed by proliferating B cells within active germinal centers, our results suggest that LN CD4(+)PD1(hi) T cells likely localize to active GC regions, a characteristic that is attributable to Tfh cells. Overall, our findings suggest that high levels of PD-1 expression on CD4(+) T cells in LN of rhesus macaques can serve as a valuable marker to identify Tfh cells and has implications for studying the role of Tfh cells in Human immunodeficiency virus (HIV), Simian immunodeficiency virus (SIV) and other infectious diseases that use the rhesus macaque model.