Serotonin N-acetyltransferase (SNAT) is a crucial enzyme in the melatonin synthesis pathway, playing an essential role in both melatonin biosynthesis and plant resistance to abiotic stress. A bioinformatics approach was employed to identify the members of the citrus SNAT gene family and to analyze their physicochemical properties, gene structure, conserved domains, phylogenetic relationships, and promoter cis-acting elements. Additionally, the tissue-specific expression of trifoliate orange SNAT family members and their expression patterns under stress conditions were examined. This study identified 21 members of the SNAT gene family across five citrus genomes, distributed over five chromosomes, with the majority predicted to localize within chloroplasts. These genes were characterized by having between 1 and 8 exons, 0 and 7 introns, 1 and 2 conserved domains, and 5 and 8 motifs. Phylogenetic analysis classified the genes into four subgroups, demonstrating significant collinearity with SNAT genes in rice. Analysis of the promoter regions revealed 32 cis-acting elements, with those responsive to light, abscisic acid, and drought being the most common. Expression analysis of SNAT genes in trifoliate orange indicated tissue specificity, with the highest expression levels detected in leaves. Quantitative real-time PCR analysis showed that the PtrSNAT1 gene was notably upregulated under various stress conditions, suggesting its role in stress response. Overall, these findings provide critical insights for further functional studies of citrus SNAT genes in relation to abiotic stress responses. Moreover, the PtrSNAT1 gene represents a potential target for developing rootstocks with enhanced resistance to abiotic stress.