Screening for pathogenic Vibrio parahaemolyticus has become routine in certain areas associated with food-borne outbreaks. This study is an evaluation of the CHROMagar Vibrio (CV) medium-PCR protocol and the conventional method (TCBS (thiosulfate-citrate-bile salts-sucrose) agar plus biochemical and Wagatsuma agar tests) for detection of V. parahaemolyticus in shrimp, water, sediment, and stool samples collected for biosurveillance in an endemic area of northwestern Mexico. A total of 131 environmental and clinical samples were evaluated. The CV medium-PCR protocol showed a significantly improved ability (P < 0.05) to isolate and detect V. parahaemolyticus, identifying isolates of this bacteria missed by the conventional method. Although some other bacteria, distinct from pathogenic V. parahaemolyticus, produced violet colonies similar to that of V. parahaemolyticus on CV medium, we were able to detect a superior number of samples of V. parahaemolyticus with the CV medium-PCR protocol than with the conventional method. The Kanagawa phenomenon is routinely determined on Wagatsuma agar for the diagnosis of V. parahaemolyticus (pathogenic) positive for thermostable direct hemolysin (TDH) in developing countries. In our results, Wagatsuma agar showed low sensitivity (65.4% at 24 h and 75.6% at 48 h) and specificity (52.4% at 48 h) for identifying V. parahaemolyticus positive for TDH. Overall, our data support the use of the CV medium-PCR protocol in place of the conventional method (TCBS-biochemical tests-Wagatsuma agar) for detection of pathogenic V. parahaemolyticus, both in terms of effectiveness and cost efficiency.