SPX (SYG1/Pho81/XPR1) family genes play a pivotal role in phosphorus signaling, phosphorus uptake, and phosphorus translocation in plants. However, to date, the SPX family genes have not been systematically investigated in cotton. In this study, we conducted a genome-wide analysis and identified 44 SPX family genes in Gossypium hirsutum, classifying them into four subfamilies (SPX, SPX-MFS, SPX-EXS, and SPX-RING) based on conserved domains. An expression analysis revealed that the majority of SPX family genes were highly expressed in the root and stem. We identified hormone response, stress response, low-temperature response, and PHR1 binding sequence (P1BS) cis-elements in the promoters of the SPX genes. Additionally, the expression of GhPHO1-4, GhSPX1-1/1-2/1-3, and GhSPX-MFS2-1/2-2 was significantly altered under phosphorus-deficient conditions and may be involved in the regulation of Pi response. A Y2H assay suggested that GhSPX1-1 interacts with GhPHR1A and GhSPX1-2 interacts with GhPHL1A. Our findings provide a basis for further cloning and functional verification of genes related to the regulatory network of low phosphorus tolerance in cotton.