In controlled environments, hydraulic conductance of cotton (Gossypium hirsutum L.) roots is affected by nitrate supply. Limited information is available on the influence of nitrogen (N) fertilizer application on cotton root hydraulic conductance under field conditions. The objective of this study was to determine if applied N influenced root hydraulic conductance and lint yield of four diverse genotypes under field conditions. Studies were conducted in 2009 and 2010 at Florence, SC and Stoneville MS. Treatments were two applied N fertilizer rates (0 and 112 kg N ha-1) and four genotypes (AGC 85, PD 2, Siokra L23, and Tamcot 22). Root hydraulic conductance was measured twice each year at Florence and once each year at Stoneville. Nitrate-N (NO3-N) was determined in the stems of the plants on which root hydraulic conductance was measured. Cotton yield was measured at the end of the season. Stem NO3-N concentration was higher in the N-fertilized cotton plant stems than in the stems of the unfertilized plants at all measurement times. Nitrogen fertilizer increased yield by 47% at Florence and by 23% at Stoneville. An N rate X genotype interaction occurred for lint yield at Stoneville. Nitrogen fertilization significantly increased lint yield for AGC 85, Siokra L23, and Tamcot 22 but not for PD 2. No significant interaction between N rates and genotypes occurred at Florence. All four genotypes had similar increases in boll weight and decreases in lint percent with N fertilization, suggesting that the differential yield increase among genotypes with N was due to boll number and not yield components. No differences occurred between N rates or among genotypes for root hydraulic conductance.