The present study was conducted to evaluate genetic analysis of fuzzless seed trait in cotton. One hundred and two upland cotton (G. hirsutum) and eighty-five island cotton (G. barbadense) were used to cross with the same lines, TM-1 (G. hirsutum) and Xinhai 13 (G. barbadense), respectively. Two different F1 populations obtained were assessed to specify the dominant and recessive inheritance of fiber fuzziness in these lines. Three F1 populations (Kuguangzi × TM-1, Luwuxu × TM-1, and SA65 × TM-1) displaying recessive fiber fuzziness inheritance were selected to construct the F2 population for a further genetic study of fuzzless seed trait. The results of this study indicated that (1) the same materials showed different quantities of fuzzy fiber in different environments. Less fuzzy fiber was found in Xinjian and Hainan compared to Anyang. Thus, the quantity of fuzzy cotton seed depends on ecological environment. (2) In upland cotton, the inheritance of fiber fuzziness was dominant for 26 accessions (25.49%), incompletely dominant for 8 accessions (7.84%), and recessive for 22 accessions (21.57%). The inheritance of fiber fuzziness in island cotton was dominant for 5 accessions (5.88%), incompletely dominant for 16 accessions (18.82%), and recessive for 9 accessions (10.59%). Analysis of F2 population indicated that the fiber fuzziness of Kuguangzi was controlled by two recessive complementary effect alleles. The fiber fuzziness of Luwuxu was controlled by two recessive additive effect alleles, and a single recessive gene controlled the same trait for SA65. Fiber fuzziness evaluation in cotton germplasm provides the genetic and basic information for cotton fiber development study and breeding.