Technical Abstract: Two of the primary impeding forces in the genetic improvement of cotton are: 1) the lack of information about genes that control important traits and 2) lack of an easy way to use diverse germplasm. Chromosome substitution lines can be useful resources to provide comprehensive genomic information of important fiber and agronomic traits and to enhance germplasm of Upland cotton. Recently we have developed a series of backcrossed chromosome substitution lines (CS-B) with a homologous pair of chromosome or chromosome arms of G. barbadense (3-79) germplasm substituted for the homologous G. hirsutum (TM-1) chromosomes or chromosome segments. The backcrossed chromosome substitution lines are genetically similar to TM-1, an Upland cotton (G. hirsutum) genetic standard, and to each other, except that each line differs by the replacement of a specific homologous pair of chromosomes or chromosome segments from the donor alien species. The overall goal of this paper is to report on agronomic and fiber trait performance of four euploid (2n=52) backcrossed chromosome or chromosome arm substitution lines including chromosomes 01, 11sh (chromosome 11 short arm), 12 sh, and 26 Lo (chromosome 26 long arm). Data for yield, boll size, lint percentage, micronaire, 2.5% span length, elongation, and strength were collected from four replicated field experiments at two different locations in two years and analyzed under an additive dominance (AD) genetic model with genotype and environment interaction. Our chromosomal assignment of effects generally agreed with published map information. CS-B 12sh had higher, while CS-B 01 and CS-B 26Lo had lower boll weight than TM-1. Results showed that CS-B 26Lo had significant negative homozygous dominance effect, but high positive heterozygous dominance effect on seedcotton and lint yields. The presence of significant negative additive effects for micronaire with CS-B 01 and significant positive additive effects for elongation and fiber strength with CS-B11sh suggested the substituted chromosome arms of 3-79 in these CS-B lines were more likely carrying genes causing these effects. CS-B lines provided valuable information on the effects of an alien species chromosome or chromosome arm on fiber and agronomic traits in Upland cotton. These CS-B lines also provide novel genetic resources for improving Upland cotton germplasm.