Sub-clinical mastitis considered as a leading hindrance in the development of dairy industry in Pakistan. Pakistan is the top listed country with the highest prevalence of Sub-clinical mastitis. Therefore, this study was conducted to estimate the prevalence and assess the potential risk factors of sub-clinical mastitis in lactating buffaloes in Pothohar region of Pakistan. A questionnaire based cross-sectional based survey was conducted in 30 commercial and subsistent farms in Pothohar region of Rawalpindi district, Punjab. Where randomly 196 lactating buffaloes were sampled. Data on several health, management and bio-security variables was collected in face to face interviews from farmers, managers and owners. Milk samples collected were subjected to California Mastitis Test.Data entry and validation was performed through Epi-Data. Data analysis was performed through SPSS. Chi-square and regression analysis were conducted. An overall prevalence of 67.3% was found. On multivariable logistic regression several health (lactation stage, number of lactations, body mass index, udder shape and milk yield), management (udder preparation and teat dipping) and bio-security (source of animal, feed sharing, manure removal and de-worming) factors were identified as potential risk factors (OR>1; P-Value<0.05). An increasing trend of prevalence of sub-clinical mastitis was observed considering previous studies from Pakistan. It can be concluded that the health, management and bio-security measures are under satisfactory level. To control the epidemic trend of sub-clinical mastitis in Pakistan addressing these factors needs to be prioritized.