The most significant traits in agricultural biotechnology are those resulting from expression of transgenes, introduced to plant species such as cotton which has incorporated genes encoding resistance against biotic and abiotic stress. This chapter emphasizes on characterization of transgenic cotton plants in terms of polymerase chain reaction (PCR) techniques. This chapter describes in detail the protocol for primer design, genomic DNA and total RNA extraction, cDNA synthesis from RNA, master mix preparation, thermo cycler procedure, and finally analysis of PCR amplified product by agarose gel electrophoresis. This chapter describes not only the basic foundations of PCR, for instance, primer design, thermal cycling, and DNA amplification. It also covers the aspect of using reverse transcription PCR (RT-PCR) which is a type to measure gene expression amounts by transcribing RNA as complementary DNA (cDNA) before amplification. One needs to use RT-PCR for a more functional readout, especially if trying to measure the change caused by the transgene insertions.