The DNAs of two diploid species of Gossypium, G. herbaceum var. africanum (A1 genome) and G. raimondii (D5 genome), and the allotetraploid species, G. hirsutum (Ah and Dh genomes), were characterized by kinetic analyses of single copy and repetitive sequences. Estimated haploid genome sizes of A1 and D5 were 1.04 pg and 0.68 pg, respectively, in approximate agreement with cytological observations that A genome chromosomes are about twice the size of D genome chromosomes. This differences in genome size was accounted for entirely by differences in the major repetitive fraction (0.56 pg versus 0.20 pg), as single copy fractions of the two genomes were essentially identical (0.41 pg for A1 and 0.43 pg for D5). Kinetic analyses and thermal denaturation measurements of single copy duplexes from reciprocal intergenomic hybridizations showed considerable sequence similarity between A1 and D5 genomes (77% duplex formation with an average thermal depression of 6 Β°C). Moreover, little sequence divergence was detectable between diploid single copy sequences and their corresponding genomes in the allotetraploid, consistent with previous chromosome pairing observations in interspecific F1 hybrids.