The Salt Overly Sensitive (SOS) pathway is a crucial ion homeostasis process in crop plants trafficking excess Na+ ions for elimination/sequestration. The SOS pathway genes SOS1 (Na+ /H+ antiporter), SOS2 (CIPK), and SOS3 (CBL) associated with ion homeostasis were isolated and characterized in the sugarcane clone Co 85019. The isolated genes had a coding region of 1086, 904, and 636 bp, respectively. A nucleotide blast analysis of the isolated SOS gene sequences showed strong similarity with previous genes found to be involved in the active functioning of the SOS pathway for ion homeostasis conferring salinity tolerance in sugarcane. The analysis of tissue specific gene expression of the identified SOS genes revealed a significant linear increase in the leaves under the first 96 h of salt stress (2.5- to 21.6-fold) in the tolerant genotype Co 85019, while the expression in the roots showed a linear increase up to 48 h and thereafter a gradual decline. The expression of SOS genes in the susceptible genotype (Co 97010) was significantly lower than in the tolerant genotype. Tissue ion content analysis also revealed a differential accumulation of Na+ and K+ ions in the contrasting sugarcane genotypes (Co 85019 and Co 97010) and this corroborates the varied expressions of SOS genes between the tolerant and susceptible varieties under salinity. Genome-wide analysis of identified SOS family genes showed the homologs in Saccharum complex members, Sorghum bicolor and Zea mays, and this verifies a close genetic similarity among these genera.