Experimental advanced-generation backcross populations contain individuals with genomic compositions similar to those resulting from interspecific hybridization in nature. By applying a detailed restriction fragment length polymorphism (RFLP) map to 3662 BC3F2 plants derived from 24 different BC1 individuals of a cross between Gossypium hirsutum and G. barbadense, large and widespread deficiencies of donor (G. barbadense) chromatin were found, and seven independent chromosomal regions were entirely absent. This skewed chromatin transmission is best accounted for by multilocus epistatic interactions affecting chromatin transmission. The observed frequencies of two-locus genotypes were significantly different from Mendelian expectations about 26 times more often than could be explained by chance (P < or = 0.01). For identical pairs of loci, different two-locus genotypes occurred in excess in different BC3 families, implying the existence of higher-order interlocus interactions beyond the resolution of these data. Some G. barbadense markers occurred more frequently than expected by chance, indicating that genomic interactions do not always favor host chromatin. A preponderance of interspecific allelic interactions involved one locus each in the two different subgenomes of (allotetraploid) Gossypium, thus supporting several other lines of evidence suggesting that intersubgenomic interactions contribute to unique features that distinguish tetraploid cotton from its diploid ancestors.