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Modernization and the global fashion market demand continuous improvements in upland cotton cultivars (<i>Gossypium hirustum</i> L.) to meet these improved fiber characteristics (fiber length, fiber strength, micronaire) requirements. Researchers have centered their efforts on improved fiber quality; however, the efforts are not immediately supporting the textile sector. The daily mean and temperature amplitude fluctuation affect cotton yield and fiber characteristics. This study analyzed four newly developed cotton varieties in two cotton regions for fiber characteristics’ variations. It was observed that cotton fiber quality characteristics (fiber length, uniformity, strength, and micronaire) are impacted in diverse ways. Fiber quality is mainly affected by the genotype and environmental conditions, e.g., weather conditions, irrigation management, fertilization, and cultural practices. The Khanewal region had shown better fiber characteristics than the Multan region, whereas cotton variety CIM-785 had better fiber characteristics in both regions.