Objective: To study the relationship between drug resistant genetic mutation and drug resistance in Mycobacterium tuberculosis L-form, discuss the internal relationship between drug resistances and drug-resistant related genes and explore the value of PCR-SSCP to clinical application. Methods: A total of 52 clinically isolated strains of tuberculosis L-form were collected among 97 pneumoconiosis patients complicated with tuberculosis. The gene mutations of katG, rpoB and rpsL were detected by PCR-SSCP, and the results were compared with those analyzed by traditional antimicrobial susceptibility test(AST). Results: The gene mutation rates of katG, rpoB and rpsL by PCR-SSCP were respectively 57.70%(30/52), 65.38%(32/52) and 40.38%(21/52). The rate of reversion was 78.85%(41/52) and the result of drug-resistant genes was invariable. The results of AST showed that there were 40(76.92%) multi-drug resistant strains in 52 clinically isolated strains. The number for three-drug resistant strain was 21(40.38%) and that of two-drug resistant was 19(36.54%), but only 12(23.08%) strains were one drug resistant. The rate of total drug-resistance was 100%, but there were 15 strains of allied mutation of three genes, 16 of two mutations and 6 of only one by PCR-SSCP. The coincidences were respectively 71.43%, 84.12% and 50.00%. Then there was no significant difference between the allied mutations of multi-drug resistant gene and the mutations of only one drug resistant gene(P > 0.05). Conclusion: PCR-SSCP technique has a higher sensibility and specificity to detect the genes of katG, rpoB and rpsL in tuberculosis L-form among pneumoconiosis complicated with tuberculosis,and the detecting rate of two drug resistant strains and three drug resistant strains was higher. The combined application of PCR-SSCP and AST has advantages at earlier diagnosis and guidance of clinical medications.