Thrips are one of the most important cotton (Gossypium spp.) insect pests in the early growing season and can cause yield losses up to 1% even after one insecticide application. Development of thrips-resistant cultivars represents the most effective strategy for control. The objective of the current study was to investigate the genetic basis of thrips resistance in susceptible Upland (Gossypium hirsutum L.) × resistant Pima (G. barbadense L.) crosses and to identify thrips-resistant lines from the interspecific hybrids. Among the five tetraploid cotton species, G. tomentosum Nutt ex Seem. with the Pilose trait was the most resistant, followed by G. mustelinum Miers ex Watt, G. barbadense, and G. darwinii G. Watt, with Upland cotton being the most susceptible. In 14 F2 populations of four Pima × four Upland crosses, segregation of thrips resistance in seven populations followed a 3 resistant:1 susceptible ratio, indicating a major dominant thrips-resistance gene (tentatively named as Thr) in the Pima parents. Among 146 backcross inbred lines derived from Upland Sure-Grow (SG) 747 × Pima S-7 and 90 recombinant inbred lines derived from Acala 1517-99 × Pima Phy 76, more than 30 lines displayed thrips resistance similar to the Pima parent. This indicates that the thrips resistance in Pima cotton can be successfully transferred into Upland cotton through backcrossing or pedigree selection. Broad-sense heritability estimates for thrips-resistance evaluations in the greenhouse ranged from 0.68 to 0.79, with at least one resistance gene estimated, indicating that the majority of the variation in thrips resistance is determined by genetic factors.