NDR1/HIN1-like (NHL) genes play essential roles in triggering plant defenses in response to environmental stimuli. In the present study, we performed comprehensive identification, characterization, and transcription analysis of NHL genes in Glycine max under chilling stress. Phylogenetic analysis showed that the 36 GmNHLs could be divided into 4 subgroups. Cis-element analysis revealed that the majority of their promoter regions contain phytohormone-responsive and abiotic stress-responsive elements, indicating their potential functions in stress responses. Germination analysis of soybean seeds resulted in detection of significantly delayed germination under chilling stress (10 ℃) and with abscisic acid (ABA, 15 μM) treatment; transcription analysis revealed GmNHLs to be differentially regulated in response to chilling stress and ABA treatment. In particular, NP_001239877.1 and XP_003532975.1 were significantly up-regulated, indicating a potential role of GmNHLs in mediating the crosstalk of responses to these treatments. In addition, key ABA biosynthesis genes and signal transduction genes were up-regulated under chilling stress. Therefore, we suggest that GmNHLs might be involved in regulating seed germination under chilling stress through the ABA regulation pathway.