<p>Wet markets have been implicated in the outbreak of recent pandemics like Severe Acute Respiratory Syndrome (SARS), H5N1 avian influenza, and most recently, COVID-19. In view of the wet markets as a potential source of spillover, our study aimed to assess hygiene, sanitation, and biosecurity practices in wet markets across Punjab, a state in India. A total of 60 wet market stalls from three different regions of Punjab were subjected to analysis through participatory and observational survey using 45 questions covering 12 parameters. A quantitative assessment scoring system was developed as per the guidelines of World Health Organization (WHO) which was used to evaluate the wet market stalls. Among the 60 wet market stalls that were assessed, 13 (21.67%) of them were categorized as having poor hygiene, sanitation and biosecurity practices. 39 (65%) stalls were classified as having moderate practices, and 8 (13.33%) stalls were observed to follow good hygiene and biosecurity measures. None of the stalls were found to have excellent practices, emphasizing the need for continuous efforts to elevate hygiene and biosecurity standards in these wet markets. Educational campaigns and agency coordination, such as with Food Safety and Standards Authority of India (FSSAI) and municipal bodies, are essential for licensing wet market stalls. Emphasizing cleanliness, protective gear provision, and zoning can improve hygiene and reduce disease risks, protecting public health.</p>