As an important crop for US agricultural security and sustainable agriculture, cotton genetic improvement requires development of innovative analytical tools. Genetic analyses based on individual gametes provide several advantages including minimum sample requirement, genetic identification of male parent and overcoming the complexity of polyploidy due to haploid nature of microspores. This research used tetraploid cotton (G. hirsutum x G. barbadense) chromosome substitution (CS) lines with G. barbadense chromosomes 17 and 25 in G. hirsutum background. The individually isolated pollen grains were germinated to release their DNAs and genomic DNA was increased by modified primer extension pre-amplification (PEP) using MasterAmp™ Extra-Long PCR Kit (EPICENTRE®, Madison, WI). Also from select cotton lines, microspores just released from tetrad developmental stage were individually isolated and gamete DNA was extracted as well as amplified through multiple displacement amplification (MDA) using REPLI-g Single Cell Kit (QIAGEN, Valencia, CA). The parental samples along with PEP and MDA amplified individual gamete DNAs were then analyzed using simple sequence repeat (SSR) as well as IRD800 and IRD-700 labeled (Li-Cor, Lincoln, NE) amplified fragment length polymorphism (AFLP) methods. Nineteen SSR and 28 AFLP primer pairs were used to analyze TM-1, 3-79, CS-B17 and CS-B25 cotton lines. The amplification of parental SSR and AFLP markers from both mature pollen as well as early free microspore samples provided unique tools for comparative genetic studies using agarose and polyacrylamide gel electrophoresis, respectively.