Fifty-four isolates of Vibrio parahaemolyticus were examined for hemolytic and urease-producing phenotypes as well as presence of virulence markers by polymerase chain reaction (PCR). All clinical isolates (11/11, 100%) and one out of 42 isolates from seafood (2.4%) possessed the tdh gene and showed hemolysis. This tdh-positive seafood isolate as well as four clinical isolates belonged to the new pandemic clone O3:K6 according to serotyping and sequencing of the toxRS locus. The new O3:K6 clone, O1:KUT, and O4:K68 shared the same characteristic variations from the old O3:K6 clone at six base positions from 576 to 1244 of the toxRS locus. Seven clinical isolates (63.6%) were positive on both toxRS- and orf8-based PCRs. Our results indicate that the new pandemic O3:K6 clonal group of V. parahaemolyticus isolates from clinical and seafood sources are present in the southeastern coastal Chinese province Zhejiang and the pandemic clones of V. parahaemolyticus should be targeted for control of seafood-related transmission to humans.