Molecular typing of Vibrio cholerae strains is a powerful tool for the surveillance of cholera. Amplified fragment length polymorphism (AFLP) is considered to be a powerful subtyping technique to distinguish bacterial strains at the genetic level. Optimization and standardization of AFLP protocol is required to allow data comparisons across different laboratories in a surveillance network. Here, we performed AFLP using different restriction enzymes and primer pairs for subtyping of V. cholerae serogroups O1 and O139 and compared the optimized AFLP protocol with pulsed-field gel electrophoresis (PFGE) to evaluate the applicability of AFLP for conducting epidemiological surveillance of cholera. The discriminatory index (D-value) of PFGE for serogroup O1 strains was similar when digested with NotI and SfiI, whereas that for O139 strains was higher for NotI digestion than for SfiI. EcoRI-G/MseI-T was the restriction enzyme and primer combination with highest discriminatory index used in the AFLP analysis. Capillary electrophoresis-based AFLP showed higher discriminatory power than that of polyacrylamide gel electrophoresis-based AFLP. When the two methods were compared using 72 epidemiologically unrelated serogroup O1 El Tor isolates, AFLP had a lower D-value than PFGE with NotI and SfiI digestions, respectively. For 54 epidemiologically unrelated serogroup O139 isolates, NotI PFGE had the highest discriminatory power, and SfiI PFGE and AFLP yielded almost the same but lower discriminatory power. We conclude that NotI and SfiI are both suitable for the PFGE of V. cholerae serogroup O1, whereas NotI should be defined as the primary enzyme for serogroup O139. The applicability of AFLP in V. cholerae subtyping and outbreak investigations is limited.