In plants, the basic helix-loop-helix (bHLH) transcription factors play key roles in diverse biological processes. Genome-wide comprehensive and systematic analyses of bHLH proteins have been well conducted in Arabidopsis (AtbHLH), rice (OsbHLH) and tomato (SlbHLH) species. However, there are no reports of bHLH family genes have been functionally characterized in Persian walnut (Juglans regia). We identified a total of 189 bHLH genes in J. regia, which are clustering into 18 subfamilies. All JrbHLH contained the conserved domains HLH. A total of 69 JrbHLH genes were expanded as whole genome duplications (WGD). The syntenic analysis was conducted for Persian walnut (J. regia) with other two woody plants (P. trichocarpa and O. europaea) to infer the bHLH genes’ evolutionary relationship between these species .We identified a total of 42 pairs of orthologous bHLH genes between J. regia and P. trichocarpa, while only 29 orthologous gene pairs between J. regia and O. europaea, while 25 collinear gene pairs were found in three species J. regia, O. europaea and P. trichocarpa. Furthermore, JrbHLHs had different expressed patterns between reproduction and vegetative tissues based on the transcriptome expression profiles of female flowers, male flowers, leaves, and hulls, respectively. Specifically, both transcriptome data and quantitative polymerase chain reaction (qRTPCR) assessment showed that two JrbHLH genes (JrbHLH3-5 and JrbHLH13-13) were highly represented in female and male flowers, while one JrbHLH gene (JrbHLH13-4) was highly expressed in leaves. Genome-wide identification, gene structure, phylogeny and expression analysis of the JrbHLH gene in J.regia, supplied a theoretical foundation for the functional study of the JrbHLH gene family and provide an understanding of the further facilitated molecular studies of Persian walnuts.