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We establish the presence of Vibrio parahaemolyticus and deepen the comparison of isolates using MALDI-TOF MS for the typing of isolates originating from the Khnifiss lagoon (Morocco). Amongst 48 samples from sea water, sediment and shellfish isolated from different sites of Khnifiss lagoon, Morocco, we obtained 22 isolates of V. parahaemolyticus identified by Vitek 2™ System (bioMérieux) and MALDI Biotyper™ (Bruker Daltonics). All isolates were highly resistant to ampicillin and ticarcillin, moderately resistant to cefalotin, but sensitive to 16 other antimicrobials tested. MALDI-TOF MS was used to discriminate between closely related environmental strains of V. parahaemolyticus. A clustering and distribution based on MALDI-TOF spectra were generated using the BioTyper 1.1™ software. Despite low diversity in regard to the biochemical characteristics and antimicrobial resistance, the isolates evoke a larger biodiversity when analysed through mass spectra of abundant proteins. Different evaluations of a cut-off value showed that, when placed at a 10% threshold of the whole diversity, isolates differed by at least three mass peaks.This work establishes the presence of Vibrio paraheamolyticus in the Khnifiss lagoon (Morocco), which has international importance for both environmental, fishing and tourism activities. Bacterial identification through Vitek 2® and the MALDI-TOF Biotyper® system was consistent. Twenty-two isolates were assessed by mass spectrometry, and the isolates were classified on the basis of constant peptide peaks and their intensity. According to peak intensity, a cut-off of dissimilarity significance is presented.