NR2A and NR2B are the major GluR2 subunits of N-methyl-D-aspartate (NMDA) receptor. NR2B-containing NMDA receptor was found to be exclusively expressed in post-synapses in layer III pyramidal cells of the prefrontal cortex (PFC). Many studies have suggested the importance of PFC NR2B-containing NMDA receptor for working memory, especially for the persistent delay cell firing. However, direct evidence for the necessity of PFC NR2B-containing NMDA receptor on working memory is still absent, especially in non-human primates. Here, we directly evaluated the necessity of PFC synaptic NR2B for working memory in both rats and monkeys. We first examined the synaptosome expression ratio of NR2B/2A in the PFC, hippocampus and visual cortex, and confirmed a relatively higher expression ratio of NR2B/2A in the PFC than in the hippocampus and visual cortex in both species. We then investigated the effects of intra-PFC blockade of NR2B on the performance of spatial working memory and pattern discrimination, and found that the spatial working memory, but not pattern discrimination, was robustly impaired in a delay length-dependent way in both species. The present study provided behavioral and neuropharmacological evidence for the critical role of PFC NR2B-containing receptors in working memory performance in non-human primates.