No study has determined what proportion of the total microbiota comprises the genus Mycobacterium in ovine Johne’s disease (OJD) tissues. We aimed to assess the relative abundance of Mycobacterium in the ileocaecal lymph node, involved and uninvolved ileal mucosa from sheep with and without OJD, using three extraction methods. Eight sheep, four with and four without OJD, were recruited. Pyrosequencing of the 16Sr RNA gene amplicons for all samples revealed that Mycobacterium represented between 0-92% (average 38%) of the total microbiota of samples from sheep with OJD, and 0-85% (average 13%) of sheep without OJD. Only sheep with OJD had samples that were positive for the IS900 (MAP) element. Mycobacterial strains other than MAP may provide competitive exclusion of MAP and should be further investigated.