Abstract: To accurately analyze the resistance of different tobacco varieties (lines) to Meloidogyne incognita, a pot experiment was conducted to compare differences in plant height, stem circumference, root fresh mass, stem fresh mass, and leaf fresh mass among 20 varieties (lines) under M. incognita inoculation. Principal component analysis (PCA), membership function analysis, cluster analysis, and stepwise regression analysis were combined to comprehensively evaluate the resistance of different tobacco varieties (lines) to M. incognita. The results showed that the disease resistance coefficients of the five agronomic traits were significantly or extremely significantly correlated with one another under M. incognita treatment. Through PCA, the five individual traits were transformed into two independent comprehensive indicators, with contribution rates of 49.948% and 22.685%, respectively, collectively explaining 72.633% of the total variance. Based on the comprehensive resistance evaluation value (D) calculated using the membership function method, cluster analysis divided the 20 varieties (lines) into three resistance types: resistant (4 varieties), moderately resistant (10 varieties), and susceptible (6 varieties). A stepwise regression equation was established as D(X) = −1.432 + 0.504X₁ + 0.589X₃ + 0.661X₄ + 0.719X₅ (R² = 0.992), which could accurately predict the resistance levels of different varieties (lines). This study demonstrates that using plant height, root fresh mass, stem fresh mass, and leaf fresh mass as key indicators, combined with multivariate statistical analysis methods, is feasible for evaluating tobacco resistance to M. incognita, and the 20 tobacco varieties (lines) could be classified into three resistance categories accordingly.