The N‐methyl‐D‐aspartate receptor (NMDA‐R) has been inter alia implicated in synaptic plasticity, brain development and emotional processes. The NMDA‐R is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. We generated NR2C‐2B mutant mice in which an insertion of NR2B cDNA into the gene locus of the NR2C gene replaced NR2C by NR2B expression throughout the brain. This NR2C‐2B mutant was used to examine whether an NMDA‐R subunit exchange in juvenile mice would affect emotional behaviors and acetylcholine (ACh), dopamine (DA) and serotonin (5‐HT) content in the frontal cortex (FC) and brain structures, which are part of the brain defense system, such as the periaqueductal grey matter (PAG). Juvenile, 1‐month‐old NR2C‐2B mice showed increased open arm avoidance in the elevated plus‐maze and increased fear‐induced immobility. In terms of brain neurochemistry, NR2C‐2B mice showed an increase in 5‐HT levels in the FC at the age of 2 months. A correlational analysis revealed that mice with low open arms avoidance had high levels of ACh in the PAG but reduced 5‐HT levels in the FC. Animals which showed high levels of fear‐induced immobility also had high levels of 5‐HT in the FC. These results suggest that the replacement of subunit NR2C by NR2B in juvenile mice increases anxiety‐ and fear‐related behaviors possibly due to changes in FC‐5‐HT and PAG‐ACh levels.