Surveillance and monitoring are important for measuring the level of disease in a population, documenting changes in prevalence over time, determining high-risk areas for implementation of control measures, eradicating disease, and documenting freedom from disease. The documentation of freedom from disease has importance for international trade and the resumption of production after an outbreak. Johne's disease (JD) is an example of an endemic disease of cattle that has variable prevalence related to environmental and animal-level factors. Three methods of sample collection were used to describe the prevalence and distribution of JD seropositivity in Texas. Sampled cattle were: (1) extensively managed herds, (2) market cattle, and (3) clinically ill cattle examined by practicing veterinarians throughout Texas. Samples were evaluated for JD using a commercially available serum ELISA. Proportion of seropositive samples was compared and spatial distributions were evaluated for clustering. Difference of JD seropositivity was observed among the three sample populations suggesting that estimation of disease prevalence is dependant upon the source of samples.