The gamma-interferon assay (IFNγ) is often used as an ancillary diagnostic test alongside the tuberculin skin test in order to detect Mycobacterium bovis infected cattle. The performance of the IFNγ test has been evaluated in many countries worldwide and wider usage as a disease surveillance tool is constrained due to the relatively low and inconsistent specificity at a herd and area level. This results in disclosure of a higher proportion of false positive reactors when compared with the skin test. In this study, we used cohorts of animals from low prevalence tuberculosis herds (n = 136) to assess a range of risk factors that might influence the specificity of the test. Univariate and multivariate logistic generalised estimating-equation (GEE) models were used to evaluate potential risk factors associated with a false positive IFNγ test result. In these herds, the univariate model revealed that the region of herd origin, the time of year when the testing was carried out, and the age of the animal were all significant risk factors. In the final multivariate models only animal age and region of herd origin were found to be significant risk factors. A high proportion of herds with multiple IFNγ false positive animals were located in one county, with evidence of within-herd clustering, suggesting a localised source of non-specific sensitization. Knowledge of the underlying factors influencing the IFNγ test specificity could be used to optimize the test performance in different disease level scenarios in order to reduce the disclosure rate of false positive reactors.