Cotton is the world’s leading natural fiber and second most important oilseed crop and has been a focus of genetic, systematic and breeding research. The genetic and physiological bases of some important agronomic traits in cotton were investigated by QTL mapping through constructing of genetic map with chromosomal assignment. A segregating F2 population derived from an interspecific cross (G. barbadense x G. hirsutum) between two genotypes, cvs. “Giza 83” and “Deltapine” was used in this study. Different molecular markers including SSR, EST, EST-SSR, AFLP and RAPD were employed to identify markers that reveal differences between the parents. In total 42 new markers were merged with 140 previously mapped markers to produce a new map with 182 loci covering a total length of 2370.5 cM. Among these new markers, some of them were used to assign chromosomes to the produced 26 linkage groups. The LG2, LG3, LG11 and LG26 were assigned to chromosomes 1, 6, 5 and 20 respectively. Single point analysis was used to identify genomic regions controlling traits for plant height, number of nodes at flowering time, bolling date, days to flowering and number of bolls. In total 40 significant QTL were identified for the five traits on 11 linkage groups (1, 2, 3, 4, 5, 10, 11, 12, 18, 19 and 23). This work represents an improvement of the previously constructed genetic map in addition to chromosomal assignment and detection of new significant QTL for the five traits in Egyptian cotton. The Significant QTLs detected in this study can be employed in marker assisted selection for molecular breeding programs aiming at developing cotton cultivars with improved agronomic traits