The knowledge of the critical periods of crop-weed competition is crucial for designing weed management strategies in cropping systems. In the Lower Yangtze Valley, China, field experiments were conducted in 2011 and 2012 to study the effect of interference from mixed natural weed populations on cotton growth and yield and determine the critical period for weed control (CPWC) in direct-seeded cotton. Two treatments were applied: allowing weeds to infest the crop or keeping plots weed-free for increasing periods (0, 1, 2, 4, 6, 8, 10, 12, 14, and 20 weeks) after crop emergence. The results showed that mixed natural weed infestations led to 35 to 55 cm shorter cotton plants with stem diameters 10 to 13 mm smaller throughout the season, fitting well with modified Gompertz and logistic models, respectively. Season-long competition with weeds reduced the number of fruit branches per plant by 65% to 82%, decreasing boll number per plant by 86% to 96% and single boll weight by approximately 24%. Weed-free seed cotton yields ranged from 2,900 to 3,130 kg ha-1, while yield loss increased with the duration of weed infestation, reaching up to 83% to 96% compared with permanent weed-free plots. Modified Gompertz and logistic models were used to analyze the impact of increasing weed control duration and weed interference on relative seed cotton yield (percentage of season-long weed-free cotton), respectively. Based on a 5% yield-loss threshold, the CPWC was found to be from 145 growing degree days (GDD) to 994 GDD, corresponding to 14 days after emergence (DAE) to 85 DAE. These findings emphasize the importance of implementing effective weed control measures from 14 DAE to 85 DAE in the Lower Yangtze Valley, China, to prevent crop losses exceeding a 5% yield-loss threshold.