Background/Objectives: Cyclic nucleotide-gated channel (CNGC) genes play crucial roles in plant growth, development, and stress responses, yet their functions in sorghum remain poorly understood. Methods: This study systematically analyzed sorghum CNGC genes through genome-wide identification, encompassing chromosomal mapping, phylogenetic relationships, gene structure, cis-acting elements, miRNA regulation, and GO/KEGG annotation. Results: A total of 23 sorghum CNGC genes were identified and classified into five subclasses (I–IV-b), exhibiting high evolutionary conservation with rice and maize. Promoter and miRNA analyses revealed multi-level regulation involving light, hormones (ABA, JA), and stress response elements. Several SbCNGC genes were predicted to be regulated by multiple miRNAs. Expression profiling and qRT-PCR validation indicated that most SbCNGC genes responded to both high-temperature and low-temperature stress. Expression analysis revealed tissue specificity and stress-induced transcriptional responses. Notably, SbCNGC1 remains consistently upregulated under both cold and heat stress, suggesting a potential key role in Ca²⁺-mediated signaling. Conclusions: This study systematically characterizes SbCNGC genes for the first time, reveals their potential role in abiotic stress tolerance, and provides a valuable resource for sorghum functional genomics and molecular breeding.

Background: Sorghum (Sorghum bicolor L.) is an adversity-tolerant crop, but the function of the DUF966 gene family in its growth, development, and stress tolerance is unclear. Methods: The SbDUF966 gene was identified and analyzed using bioinformatics methods in this study. We also analyzed the expression pattern of SbDUF966 in different tissues and stress conditions using RNA-seq and RT-qPCR. We explored its post-transcriptional regulatory mechanism by combining it with miRNA analysis. Results: A total of six SbDUF966 genes were identified and categorized into two groups (Group I and Group II). Gene expression analysis showed that SbDUF966 exhibited specific expression in different tissues and developmental stages, and the expression response to abiotic stresses such as low temperature, high temperature, salinity, and flooding varied over time. In addition, 12 sorghum miRNAs were predicted as potential regulators of SbDUF966. Conclusions: The SbDUF966 gene family likely regulates sorghum’s growth, development, and stress tolerance.