The present study was aimed at developing a SYBR Green-based real-time polymerase chain reaction (PCR) assay for a rapid differentiation of the genotype G1 from the cluster of genotypes G2/G3 of Echinococcus granulosus, using as marker the 12S mtDNA gene. Eleven hydatid cysts from water buffaloes and 19 from cattle were used. Fourteen samples (identified as G1 using sequencing) showed a mean melting temperature (T (m)) of 76.4 degrees C and 16 samples (identified as G2/G3 using sequencing) showed a mean T (m) of 77.0 degrees C. The detected mean difference of the T (m) of 0.6 degrees C between G1 and G2/G3 genotypes might allow a fast and simple discrimination of these genotypes. In conclusion, the real-time PCR developed in the present study provides a powerful tool for molecular studies on E. granulosus with possibilities for extension to other genotypes using different molecular targets.