Lettuce (Lactuca sativa L.) is the most common leafy vegetable produced hydroponically in the United States. Although hydroponic systems are advantageous due to lower pest and disease pressure, and reduced water and nutrient requirements, the increasing prices of fertilizers, including phosphorus (P), still influences the profitability of hydroponic production of lettuce. Characterizing lettuce germplasm capable of producing high yield using less P inputs may help reduce fertilizer use, production costs, and P loads in wastewater. In this study, 12 lettuce accessions were grown in four experiments in a nutrient film technique system. In the first two experiments, the treatments consisted of two P concentrations (3.1 and 31 mg·L−1). Lettuce cultivated with 3.1 mg·L−1 of P had variable shoot and root biomass, root–shoot ratio, P uptake efficiency, and P utilization efficiency, indicating the existence of genetic variation. Five accessions (‘Little Gem’, 60183, ‘Valmaine’, BG19-0539, and ‘Green Lightning’) were considered efficient to P because produced similar shoot biomass with the low and high P treatments. In the third and fourth experiments, the treatments consisted of two P sources (monosodium phosphate (NaH2PO4) and tricalcium phosphate [TCP; Ca3(PO4)2]. Initially, extra 5 mM of calcium (Ca) was added to the TCP solution to reduce the TCP solubility and, hence, P bioavailability to plants. All accessions produced similar shoot and root weight with both treatments, indicating that the TCP treatment did not cause low-P stress to the plants. After, the extra Ca concentration added to TCP was increased to 10 mM, resulting in low-P stress and a significant reduction in shoot weight of all accessions. Despite the severe P stress, ‘Little Gem’ and 60183 were among the accessions with the least shoot weight reduction in the TCP treatment. Variability was observed in root biomass root–shoot ratio among accessions under the TCP treatment, suggesting that lettuce accessions responded differently to P stress conditions. The genetic variation for P use efficiency (PUE) and PUE-related traits in lettuce grown hydroponically suggests the feasibility of breeding new lettuce cultivars from elite lettuce germplasm adapted to low P availability in hydroponics.