Technical Abstract: Cotton, Gossypium hirsutum L., is an important cultivated crop that is grown throughout the world. Improvements in agronomic performance and fiber quality are needed to ensure its economic viability. Primitive accessions of cotton offer a wealth of genetic variability; however, since most of these accessions are photoperiodic they are not readily useable. This study involved 114 day-neutral derived lines that were crossed with two cultivars, Stoneville 474 and Sure-Grow 747. Day-neutral lines, cultivars, and F2-bulks were grown in field plots for two years and agronomic and fiber traits were determined. The yield for most of the F2-bulks was not greater than that of the high yield cultivars. All day-neutral lines had lint percentages that were significantly lower than the cultivars. The F2-bulks had better micronaire and fiber strength than the cultivars. Genotypic effects made significant contributions to phenotypic variation indicating diversity. Dominance effects were the primary genetic effects. No consistent patterns of collection location or taxonomic designation with genetic diversity were identified. Derived day-neutral lines provide a new source of genes for improving and expanding the genetic base of cotton.