Upland cotton, Gossypium hirsutum L., is a natural source of fiber and a major row crop in the US with an estimated $7 billion raw product value in 2019. However, it is extremely sensitive to the broadleaf herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). With the evolution of herbicide-resistant weeds compounded by off-target spray damage on conventional cotton varieties outside the transgenic Enlist technology (Dow Agrosciences) of herbicide-tolerant cotton varieties (Dow Agrosciences), there is a need to identify and develop novel sources of herbicide tolerance gene for upland cotton genetic improvement. Cotton chromosome substitution (CS) lines carry introgressions from other cultivated and wild allotetraploid Gossypium species that could be sources of novel and exotic alleles for herbicide tolerance. A total of 50 CS lines of G. barbadense L. (CS-B), G. tomentosum Nuttal ex Seeman (CS-T), and G. mustelinum Meers ex Watt (CS-M), in the genetic background of G. hirsutum L. Texas Marker-1 (TM-1) were screened for resistance to a field-recommended rate (1.12 kg ae ha-1) of 2,4-D in the greenhouse. Seven CS lines, CS-T04-15, CS-B12, CS-B15sh, CS-T04, CS-B22sh, CS-T07, and CS-B04-15 with the lowest injury were evaluated for tolerance at four and seven weeks after seedling emergence under field conditions. Progeny tests conducted in the greenhouse validated 2,4-D tolerance of CS-B15sh, showing 41% lower injury than TM-1. Novel variants of CS-T04-15 and CS-T07 were identified with complete tolerance to the herbicide but are segregating. Uptake and translocation of 14C-labeled 2,4-D indicated that reduced translocation of 2,4-D may be the 2,4-D tolerance mechanism in CS-T04-15 and CS-T07, while gene(s) associated with metabolism and reduced auxin transport appeared associated with the 2,4-D tolerance in CS-B15sh. Transcriptome analysis revealed differential expression of genes in 2,4-D-treated CS-B15sh and TM-1 with several components of the 2,4-D/auxin response pathway, including ubiquitin E3 ligase, PB1|AUX/IAA, ARF transcription factors, and F box proteins of the SCFTIR1/AFB complex being up-regulated. Functional annotation of differentially expressed genes revealed down-regulation of auxin transport, suggesting a potential linkage with tolerance mechanism involving altered movement of 2,4-D in CS-B15sh. The selected highly tolerant cotton CS lines will need to be confirmed further using molecular assays.