The cotton (Gossypium hirsutum L.) collection of primitive accessions is underutilized for cotton improvement because they are photoperiodic. Day-neutrality can be achieved by crossing with a day-neutral cultivar and selection in the F2 generation. Our objective was to determine the relationship among four backcross generations and parents using amplified fragment length polymorphism (AFLP) markers. We evaluated five populations (F6, BC1F6, BC2F6, BC3F6, BC4F6) of day-neutral plants derived from crossing and backcrossing accessions T 78, T 174, T 326, and T 1149 as recurrent parents, respectively, with 'Deltapine 16' (DPL 16) as the donor day-neutral flowering parent. Genetic distances were determined using 43 AFLP primer combinations that produced 251 polymorphic AFLP markers among the five parents and 91 to 129 polymorphic markers within each of the five populations of the four sets of crosses and backcrosses. Among the 20 backcross populations, recovery of markers from accession parents was 27 to 92%, while the recovery of markers from DPL 16 was 71 to 91%. Genetic distance of populations from the recurrent parent was 0.35 to 0.75 among backcross populations. Genetic distance from the non-recurrent parent DPL 16 was 0.16 to 0.38. Many AFLP markers tended to stay together as linked blocks and were selected with the day-neutral flowering phenotype. This finding indicates that linkage drag was occurring during introgression of the day-neutral flowering trait. These results indicate that the day-neutral populations selected after one backcross to the accession parents could be used to add genetic diversity to applied breeding programs.