Fiber mutants are important materials in genetic and functional research in cotton (Gossypium hirsutum L.). A linted-fuzzless fiber mutant, designated CM mutant, was found in transgenic cotton by Agrobacterium-mediated transformation method. The pure line of this mutant was obtained in T3 generation. PCR analysis showed that the mutated trait had no relationship with T-DNA insertion; however, point mutation during tissue culture was deduced to be responsible for the mutated trait. Analyses of inheritance and allelic tests were conducted by crossing CM mutant with TM-1, Junhai 1, and a series of fiber developmental mutants such as XinFLM, N1N1, n2n2, and T586 with linted-fuzzless fiber and XinWX, XZ142WX, SL-7-1, and MD17 with lintless-fuzzless fiber, respectively. Among the above 10 combinations, the F1 plants were fuzzless, and all the F2 generations of CM × TM-1 and CM × Junhai 1 showed the segregation ratio of 3:1 of linted-fuzzless to linted-fuzzed phenotypes. Based on the genetic analysis, we indicated that there was one dominantly different locus between the mutant and TM-1 or Junhai 1. Allelic tests and gene mapping showed that the fuzzless gene of the mutant was allelic to N1, dominantly controlling naked-seed trait. The scanning electron microscopy observation was conducted to show the development of fiber cell initials in CM mutant during early developmental stages (0–3 d post anthesis). Like N1N1 mutant, CM mutant delayed fiber cell formation and elongation under the control of mutated gene. Compared to N1N1, CM mutant exhibited significant higher lint percentage of CM and significant lower 100-seed weight. The mutated gene in CM is speculated to be one of the multiple alleles of dominant naked-seed N1 gene.