SHORT INTERNODE (SHI)-Related Sequence (SRS) transcription factors play crucial roles in plant growth, development, and stress responses and have been extensively studied in various plant species. However, the molecular functions and regulatory mechanisms of SRS genes in the economically important tropical fruit crop pitaya (Hylocereus undatus) remain poorly understood. This study identified 9 HuSRS genes in pitaya via bioinformatics analysis, with subcellular localization predicting nuclear distributions for all. Gene structure analysis showed 1–4 exons, and conserved motifs (RING-type zinc finger and IXGH domains) were shared across subclasses. Phylogenetic analysis classified the HuSRS genes into three subfamilies. Subfamily I (HuSRS1–HuSRS4) is closely related to poplar and tomato homologs and subfamily III (HuSRS6–HuSRS8) contains a recently duplicated paralogous pair (HuSRS7/HuSRS8) and shows affinity to rice SRS genes. Protein structure prediction revealed dominance of random coils, α-helices, and extended strands, with spatial similarity correlating to subfamily classification. Interaction networks showed HuSRS1, HuSRS2, HuSRS7 and HuSRS8 interact with functional proteins in transcription and hormone signaling. Promoter analysis identified abundant light/hormone/stress-responsive elements, with HuSRS5 harboring the most motifs. Transcriptome and qPCR analyses revealed spatiotemporal expression patterns: HuSRS4, HuSRS5, and HuSRS7 exhibited significantly higher expression levels in callus (WG), which may be associated with dedifferentiation capacity. In seedlings, HuSRS9 exhibited extremely high transcriptional accumulation in stem segments, while HuSRS1, HuSRS5, HuSRS7 and HuSRS8 were highly active in cotyledons. This study systematically analyzed the characteristics of the SRS gene family in pitaya, revealing its evolutionary conservation and spatio-temporal expression differences. The research results have laid a foundation for in-depth exploration of the function of the SRS gene in the tissue culture and molecular breeding of pitaya.