The chromosome substitution (CS) lines constitute an important genetic and breeding resources, increasing the genetic diversity of Upland cotton from other alien tetraploid species. Two experiments were conducted to quantify low temperature and drought stress effects during early growth stages in 21 cotton CS-lines with parent, Texas Marker (TM)-1. In Experiment I, plants were grown at optimum (30/22°C) and low (22/14°C) temperatures under optimum water and nutrient conditions. In Experiment II, plants were grown at optimum water and drought conditions. Above- and below-ground growth parameters including several root traits were assessed at 25 days after seeding. CS-lines varied significantly varied for many traits measured. Combined low temperature and drought response indices, derived from all measured parameters, showed CS-T04 and CSB08sh showed significantly higher and lower tolerance to low temperature, respectively, while CS-T04 and CS-B22sh showed significantly higher and lower tolerance to drought condition compared to TM-1.