Supplemental irrigation is known to affect the agronomic performance and fiber quality of upland cotton (Gossypium hirsutum). Enhancements resulting from supplemental irrigation, most often via improved agronomic performance, are maximized when cultivars are selected and grown that respond consistently and stably to supplemental irrigation. The objective of this study was to determine if variability exists within a subset of Pee Dee germplasm lines for their agronomic and fiber quality response to supplemental irrigation. Twelve Pee Dee germplasm lines, selected based on diverse pedigree lineage, were used in this study to provide a representation of the Pee Dee germplasm collection. This subset of Pee Dee germplasm lines, in addition to check cultivars Fibermax 966 and Stoneville 474, were evaluated under dryland and supplemental drip irrigation conditions in a split-plot design with water treatment as the main plot factor and genotype as the split-plot factor. Water treatment differences were significant for all agronomic traits measured; while differences occurred for all fiber quality traits measured except HVI uniformity, AFIS 5% span length by number, and AFIS 2.5% span length by number. Significant differences were present among Pee Dee germplasm lines for all agronomic traits measured except plant height; while differences were significant for all fiber quality traits measured except HVI micronaire, HVI uniformity, and AFIS immature fiber content. Pee Dee germplasm line response to supplemental irrigation was not different for any of the agronomic traits measured; however significant variation did exist among Pee Dee germplasm lines in response to supplemental irrigation for HVI fiber length, AFIS length by weight, AFIS upper quartile length by weight, AFIS length by number, AFIS 5% span length by number, and AFIS 2.5% span length by number. This result indicates that variation exists among Pee Dee germplasm lines for their fiber length response to supplemental irrigation.