An analytical cross-sectional study determined the serogroups and serotypes of Vibrio cholerae, and their antibiotic resistance rates, in the 2005 cholera epidemic in Hamadan. All 190 patients with positive stool cultures had V. cholerae serogroup O1, biotype El Tor and serotype Inaba positive. Of 60 cases selected randomly for antibiogram testing, sensitivity to norfloxacin, ciprofloxacin, kanamycin, amikacin, tetracycline and doxycycline was 97%, 92%, 88%, 85%, 77% and 67% respectively. Resistance to furazolidone, trimethoprim–sulfamethoxazole and erythromycin was 100%, 98% and 62% respectively. Comparison with the results of the 1998 epidemic suggests a worrying increase in the resistance of V. cholerae to erythromycin, doxycycline and ciprofloxacin.