We conducted a cross-sectional study in 2004-05 to investigate risk factors for ovine Johne's disease (OJD) involving 92 infected Merino sheep flocks in Australia. In each enrolled flock we collected pooled faecal-samples from 3-to 5-year-old sheep and cultured them for Mycobacterium avium subsp. paratuberculosis (MAP) to determine their OJD status. Based on pooled faecal-culture (PFC) results, three outcome variables representing different facets of disease biology were derived: pool OJD status (binomial: positive or negative), log pool MAP number (continuous) and cohort OJD prevalence level (ordinal: low (<2%), medium (2-10%) and high (>10%) prevalence). We used these outcomes in three separate multivariable analyses to identify risk factors, which were based on a questionnaire administered during a face-to-face interview with the farmer.We found higher OJD infection in sheep whose dams had been in poor condition and kept at a high stocking rate during lambing and in sheep which had experienced a longer period of growth retardation during their lifetime. Flocks that had vaccinated for >2 years (rather than only 1 to 2 years) with a killed MAP vaccine had significantly lower OJD infection. In addition, practices including culling low body-weight sheep or selling sub-flocks experiencing high losses, sharing of roads between neighbouring farms, and greater frequency of application of super-phosphate fertilizers were associated with higher OJD. Of the confounders investigated, infection was higher in flocks experiencing high mortalities; in wethers compared to ewes; and in 3-year-old sheep compared to 4-year-old sheep.