Introduction Clostridium perfringens presents persistent threat to the small animals in causing moderate to severe enterotoxemia. The pathogenicity of C. perfringens depends on the production of four major toxins. Therefore, typing of toxins elaborated by C. perfringens is essential. Specific toxin types are involved in causing particular enteric diseases in sheep and goats. Multiplex PCR has been introduced, which has the potential to detect the genes that code for these toxins. Also, the sensitivity and specificity of this method has been confirmed by amplification of specific target DNA under unique conditions. Therefore, the present project was designed to understand the molecular epidemiology of C. perfringens types isolated from sheep and goats. Objectives This study was carried out with following objectives: Ø To isolate, identify and characterize field strains of Clostridium perfringens isolated from sheep and goats in selected districts of Punjab, Pakistan. Ø To determine molecular epidemiology and heterogeneity of Clostridium species. Experimental Design It was an experimental study of two years duration, conducted at Multidisciplinary Research Laboratory, Isra University, Islamabad, Pakistan. vi Duration September 2013 to August 2015 Material & Methods Fecal samples were collected from healthy and diseased sheep and goats. Collected samples were cultured on cooked meat broth, blood agar and tryptose sulphite cycloserine agar. Biochemical characterization was carried out by using API 20A kits. Pure cultures of C. perfringens were used for DNA isolation by using spin column genomic DNA extraction kit. Isolated DNA was amplified by multiplex PCR with specific primers. Based on the length of the amplified fragments, bacterial strains were identified. The amplified DNA fragments were sequenced using Sanger dideoxy sequencing method. Alignment studies were carried out by using ClustalW, T-Coffee software. Results Results revealed that the major C. perfringens type among all healthy and diseased isolates was type A followed by type D. In addition to this, beta2 toxin was found in both healthy and diseased type A and D isolates. However the prevalence of beta2 toxin gene in diseased sheep and goat population was 64% as compared to 37% in healthy ones. The identified genes were found equally dispersed in both sheep and goat isolates. Nucleotide sequences of alpha, beta2 and epsilon gene revealed variations in the identified isolates. vii Conclusions It was concluded from this study that: Ø C. perfringens type A and D were prevalent in Punjab province of Pakistan while locally produced enterotoxemia vaccine did not include type A. Ø Nucleotide sequences of alpha, beta2 and epsilon gene revealed variations in the identified isolates which confirmed bacterial population heterogeneity. Ø Sequence analysis of the amplified cpb2 gene revealed two genetically different populations of the gene. Keywords Molecular epidemiology, C. perfringens, sheep, goats, multiplex PCR