Empathy is an important emotional process that involves the ability to recognize and share emotions with others. We previously developed an observational fear learning (OFL) behavioral assay to measure empathic fear in mice. In the OFL task, a mouse is conditioned for fear to the context where it observes a conspecific demonstrator receiving aversive stimuli. We have recently reported that empathic fear response is highly variable among 11 inbred mouse strains, and innate differences in conditioned fear, anxiety, locomotor activity, sociability and preference for social novelty are not significantly correlated with OFL among those strains. However, the genetic factors underlying variability in empathic fear remain to be determined. Intriguingly, we have found that mice of the 129S1/SvImJ (129S1) strain, exhibit a marked increase in OFL, as compared with another 129S substrain, 129S4/SvJaeJ (129S4). Through genetic and molecular analyses, a nonsynonymous mutation of arginine to tryptophan (R498W) in neurexin III (Nrxn3) was identified as the causative variant. This mutation occurs at a residue that is well conserved among mammalian species and is predicted to be deleterious to the protein by in silico databases. We have further determined that a deletion of Nrxn3 in the anterior cingulate cortex leads to a decrease in OFL. Knock-in mice with the R498W mutation by the CRISPR/Cas9 system are currently being tested. Taken together, we propose that Nrxn3 is an important regulator in neural circuits of OFL. These works also demonstrate the validity of the approach to utilize substrains to identify genes and alleles regulating social behaviors.