Large epidemics of diarrhoea associated with seafood consumption and Vibrio parahaemolyticus occurred during the austral summers of 2004 and 2005 in the environs of Puerto Montt, Chile (41°29′S 72°24′W). There are no reports of V. parahaemolyticus infections before 2004 in this region, their absence being explained by the low ocean temperatures which seldom reach 16°C. We analysed V. parahaemolyticus obtained from shellfish and clinical samples during epidemics. Isolates were examined using conventional protocols and an improved method for restriction enzyme analysis using total bacterial DNA which permits direct genome restriction enzyme analysis by conventional gel electrophoresis (DGREA) with a similar discrimination index as restriction fragment length polymorphism-pulsed field gel electrophoresis (RFLP-PFGE). Analysis of clinical samples showed that the epidemics were caused by the V. parahaemolyticus O3:K6 pandemic clonal group. On the other hand, analysis of shellfish samples during both epidemics showed that 53% contained V. parahaemolyticus (3–93 g−1). Detailed analysis of 50 positive shellfish samples showed that only three contained detectable levels of the pandemic clone. Most V. parahaemolyticus isolates obtained from shellfish corresponded to non-pandemic clones differentiated into 14 groups by DGREA. In summary, the causative agent during epidemics was only a minor component of a small but diverse population of V. parahaemolyticus in shellfish.