The present study aimed to utilize molecular tools to evaluate the reliability of IDEXX Paratuberculosis Screening ELISA versus traditional microscopic examination of acid fast-stained-fecal smear for rapid detection of Johne's disease among clinically suspected small ruminants in Makkah region, KSA. For this purpose, three different genetic targets, including 16S rDNA, insertion sequence 900 (IS900) and intergenic spacer (IGS), were used for molecular detection and identification of MAP among suspected cases and was used as golden standard for the evaluation study. A total of 3490 small ruminant animals from five different farms around Makkah were clinically investigated for characteristic signs of JD during the period of November 2013 to June 2014. Out of investigated animals, 41 cases were selected as being suspected of JD infection based on the associated clinical symptoms (persistent diarrhea with emaciation). Fecal samples (including rectal swabs) and blood samples were collected from all suspected small ruminants. Fecal samples were subjected to both conventional microscopic examination and molecular examination. Blood samples were used for serum separation and conduction of immunologic assay using IDEXX Paratuberculosis Screening ELISA. The results showed that out of the 41 suspected cases, 14 (34.1%) and 15 (36.6%) cases were positive for JD using microscopic examination and ELISA, respectively. On the other hand molecular evaluation of JD infection among suspected cases revealed an initial infection rate of 43.9% based on the amplification of both bacterial 16SrDNA and Mycobacterium genus-specific IGS targets. However, further investigation of suspected samples by detection of MAP-specific IS900 and sequence analysis of the Mycobacterium species-specific IGS targets confirmed MAP infection among only 34.1% of the suspected cases. Using molecular results as a standard, higher sensitivity (85.7% vs. 50%), specificity (88.9% vs. 70.4%), PPV (80% vs. 46.7%), NPV (92.3% vs. 73.1%) and AI (87.8% vs. 63.4%) were recorded for ELISA as compared to microscopic detection of AF bacilli in fecal smear, respectively. In conclusion the study revealed the feasibility of the IDEXX Paratuberculosis Screening ELISA as a reliable tool for rapid detection of Johne's diseases among suspected cases of small ruminants.