Cystinuria represents 3% of nephrolithiasis in humans with an overall prevalence of 1 in 7,000 neonates. Two genes have been reported to account for the genetic basis of cystinuria, the SLC3A1 and the SLC7A9. Recently, the possible involvement of the SLC7A10 gene in the genetic basis of the disorder was also reported. In the present study, we found a total of 15 mutations in 20 Greek cystinuric patients. Eight mutations are novel, 4 in the SLC3A1: F266S, T351I, R456C, and N516D, and 4 in the SLC7A9: 479-1G>C, Y232C, D233E, and 1399+1G>T. Furthermore, 2 polymorphisms were identified in the SLC3A1 gene and 16 polymorphic variants were also found in the SLC7A9 gene of which the 235+18C>A, 604+10G>A, and 604+24T>C are novel. Finally, no mutation was found in the SLC7A10 gene in all patients. Only, the novel 634+8C>G and the previously reported 913-11C+T polymorphisms were identified in the SLC7A10 gene. In conclusion, a spectrum of SLC3A1 and SLC7A9 mutations are responsible for the genetic basis of cystinuria in Greek patients.