Abstract: To screen safe and effective herbicides for tobacco fields and determine their optimal application rates, weed species and their infestation levels were surveyed in tobacco fields in southwestern Guizhou Province. The efficacy of eight herbicides against weeds and their safety to tobacco plants were evaluated through pre-emergence soil-sealing and post-emergence stem-leaf treatment trials. The results showed that 28 weed species belonging to 14 families were identified in tobacco fields in southwestern Guizhou, of which 8 species caused moderate to severe damage. In the pre-emergence soil-sealing trials, 35% nicosulfuron+mesotrione+atrazine OD (630.00 and 3 150.00 g a.i./hm²), 40% mesotrione SC (180.00 and 900.00 g a.i./hm²) and 48% butralin EC (1 800.00 and 9 000.00 g a.i./hm²) provided control overall plant control efficacies exceeding 89.67% against Chenopodium album, Stellaria media, Capsella bursa-pastoris, Sonchus arvensis, Galinsoga parviflora and Digitaria sanguinalis. In the post-emergence stem-leaf treatment trials, 10% glufosinate-p ammonium SL (600.00 and 3 000.00 g a.i./hm²) and 200 g/L glufosinate ammonium AS (1 800.00 and 9 000.00 g a.i./hm²) achieved fresh weight control efficacies greater than 76.24% against the same weed species. In the pre-emergence soil-sealing trials, tobacco seedling survival rates after transplanting (20 days post-treatment) were significantly reduced by 25% quinclorac SC and 40% mesotrione SC compared with the untreated control. Moreover, 40% mesotrione SC at five times the recommended dose caused complete plant death within 40 days after transplanting. At the recommended dose, all herbicides except 25% rimsulfuron WG and 48% butralin EC significantly inhibited maximum leaf width of tobacco plants. In contrast, post-emergence applications of 240 g/L clethodim EC, 48% butralin EC, 200 g/L glufosinate ammonium AS and 10% glufosinate-p ammonium SL at both recommended and five times the recommended doses promoted plant height at topping, maximum leaf length and width, and top leaf length and width. Across both pre-emergence and post-emergence trials, treatments with 25% quinclorac SC, 40% mesotrione SC and 35% nicosulfuron+mesotrione+atrazine OD induced leaf deformity or chlorosis in new tobacco leaves. Overall, 48% butralin EC, 200 g/L glufosinate ammonium AS and 10% glufosinate-p ammonium SL exhibited excellent weed control efficacy and good safety to tobacco. Butralin is recommended for pre-emergence weed control, whereas glufosinate ammonium and glufosinate-p ammonium are suitable for post-emergence inter-row weed management. Quinclorac, mesotrione and nicosulfuron+mesotrione+atrazine are not recommended for weed control in tobacco fields.