Among the wide range of antibiotic available, beta-lactam antibiotics are used widely in bacterial pathogenic infections. The most common cause of bacterial resistance against beta-lactam antibiotics is the production of beta- lactamases by many members of Enterobacteriaceae family, including espe- cially, E. col. (Harwalkar, Sataraddi et al. 2013). Extended-spectrum Î2-lacta- mases (ESBLs) are resistant to the third generation antibiotics, cephaloosporins and monobactum, additionally, these ESBL producing organismsâ TM exibit co- resistant to many other classes of antibiotics resulting limited treatment alternatives (Paterson, Bonomo 2005, Lartigue, Zinsius et al. 2007, Lewis, Herrera et al. 2007). Over the past decade, it has been observed dramatic increase of these ESBL producing E. coli strains, especially, CTX-M family of ESBL producers, to the third-generation cephalosporin antibiotic, Cefotaxime (Bonnet 2004, Lartigue, Zinsius et al. 2007, Paterson, Hujer et al. 2003, Paterson, Bonomo 2005, Ruppe, Lixandru et al. 2013). Although studies have published the diagnosis of various ESBL producers in worldwide, only few reports of molecular identification of CTX-M family of genes have been published (Al-Mayahie 2013, Harwalkar, Sataraddi et al. 2013, Literacka, Bedenic et al. 2009). This study aims to identify the DNA segments of CTX- M genes unique to E. coli, and analyse the surrounding genetic makeup of CTX-M genes. Collectively, understanding the genetic makeup of these resistance genes will shed a light on their mechanistic of antimicrobial resistance. Unlike other conventional clinical diagnostic methods, molecular diagnostics seeks evidence of a disease at the very basic causative level by detecting the nucleic acid identity. The main advantage of this diagnostic method is that the diagnosis carried out in the molecular level, and the method will identify the microbial pathogens at the early stages of the infectious diseases (Versalovic, Lupski 2002). Different types of rapid detection systems such as Real-Time PCR have been used to identify the different CTX-M family target genes (Birkett, Ludlam et al. 2007, Li, Chen 2012, Versalovic, Lupski 2002, Woodford, Fagan et al. 2006, Woodford, Sundsfjord 2005). The identified genes so far, has some degree of potency for identification, but the majority of these genes lacks the characteristics of a strong and unique genetic marker that can used as a specific target solely for identification of any CTX-M family of ESBL producing E. coli strains. Therefore this study analyse short DNA segments and develop a sensitive, specific and reliable method that can be used for rapid selective detection of ESBL producers resistant to Cefotaxime in antibiotic resistant E. coli strains.