BAC library for the Egyptian cotton Gossypium barbadense Giza 70, Giza 86, and Giza 75 varieties have been constructed and characterized. The isolation and purification of high molecular weight DNA from nuclei embedded in agarose microbeads was an essential part of this work. Several experimental parameters were investigated including optimization of megabase-size DNA restriction enzyme digest and CHEF gel conditions to achieve the highest resolution and separation of such DNA. Fragments ranging in size from 200Kb to 500Kb were size selected and recovered from agarose gel to be used in BAC library construction. The BAC vector, pBeloBAC II derived from the endogenous E.coli F-factor plasmid was used in library construction. Different insert: vector ligation ratios examined indicated 15:1 insert:vector to be the optimum ligation ratio. The maximum construct-host transformation efficiency calculated at 1.8KV electroporation voltage. Purification and isolation of BAC clones was followed by restriction enzyme digest using NotI to characterize insert sizes. Characterization of insert sizes and integrity using CHEF gel electrophoresis conditions at 6V/cm, 5 and 15 sec initial and final switch times respectively, and 12°C temperature for 12 hrs. BAC libraries for Giza 70, Giza 86, and Giza 75 varieties contained 45,237; 45,742; and 46,531 clones respectively with an average insert size of 100 Kb. Considering the haploid genome size for cotton to be 2,118 Mb, BAC libraries contains 2.12 haploid genome equivalent. Egyptian cotton BAC library constructed for Gossypium barbadense Giza 70, Giza 86, and Giza 75 varieties provide 88% probability of isolating a specific genomic region and are optimized for future gene identification, isolation, target gene mapping,and chromosome walking.