A cross-sectional study was conducted from May to September of the year 2008 in broiler flocks in the southern and northern area of Jordan, to determine the flock-level prevalence of Clostridium colinum infection. Intestinal swabs were collected from 170 broiler flocks and tested by PCR. Among the study population, 20 flocks in both areas (11.8%, 95% confidence interval: 10 to 22%) were positive for C. colinum infection. The prevalence of positive intestinal samples in the southern and northern area of Jordan were 4.7 and 7.1%, respectively, which was statistically significant (chi(2) = 3.9 df = 1, P = 0.0482). It is recommended to conduct further epidemiologic studies to determine risk factors and to evaluate the economic consequences of the C. colinum infection in the region.