Segregating populations were developed to evaluate the inheritance of the fuzzless seed phenotypes in upland cotton (Gossypium hirsutum L.). Accession 143 of the Mississippi Obsolete Variety Collection (MOVC) has a fuzzless seed phenotype. This line carries the n 2 locus which is recessive to the seed fuzz phenotype. Data from the F 2 , BC 1 F 1 , F 2 : 3 , and BC 1 F 2 populations of DP 5690 x 143 fit a two-loci model for expression of the recessive fuzzless seed phenotype. Fuzzless seeds were obtained in n 2 n 2 plants when a second recessive locus (n3) was present. The dominant N 3 allele found in DP 5690 confers the fuzzy seed phenotype in homozygous n 2 plants. Accession 243 of the MOVC carries the N 1 locus, which is dominant to the presence of seed coat fuzz. No variation from expected ratios was observed in the F 2 , BC 1 F 1 , F 2 : 3 , and BC 1 F 2 populations of the DP 5690 x 243 cross. The N 3 allele had no apparent effect on the expression of the N 1 locus. In a cross between accessions 243 x 143, a few plants were observed which were completely devoid of lint and fuzz fiber (fiberless). A fiberless line was developed from one of these fiberless plants. This line was designated MD 17 fiberless. In a cross between DP 5690 x MD 17 fiberless, we demonstrated that at least three loci were involved in the expression of the fiberless phenotype. The involvement of n 2 and n 3 in the expression of this fiberless phenotype was demonstrated in the F 2 progeny of the cross between 143 x MD 17 fiberless. This is the first demonstration that N 1 , n 2 , and n 3 interacted to produce fiberless seed.