Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that requires timely diagnosis and intervention. As frontline healthcare providers, family physicians must have a clear understanding of the diagnostic utility of rheumatoid factor (RF) and its limitations to ensure early recognition and appropriate referral of RA patients. This descriptive cross-sectional study was conducted over three months to assess the knowledge of family physicians in Punjab, Pakistan, regarding the diagnostic role and interpretation of RF in RA. Stratified random sampling was used, and a structured questionnaire was distributed to 300 physicians, with responses collected via Google Forms. The questionnaire evaluated demographic variables and knowledge of RA diagnostic criteria, RF specificity, and related clinical interpretation. The knowledge scores were classified as good (≥ 75%), moderate (50–74%), or poor (< 50%). Data analysis was performed via SPSS version 24, with chi-square tests used to examine associations (p < 0.05). The results revealed that only 27.76% of the participants demonstrated good knowledge. Most respondents had moderate (50.95%) or poor (21.29%) knowledge. Significant associations were observed between knowledge level and factors such as physician age, graduation cohort, experience, type of medical college, involvement in RA patient care, and private practice (p < 0.05). This study revealed significant gaps in family physicians’ knowledge regarding RF’s diagnostic value in RA. Targeted educational interventions and updated clinical training are essential to improve early RA detection, enhance referral practices, and improve patient outcomes at the primary care level.