ABSTRACT Digital dermatitis (DD) is an important cause of lameness in dairy cattle worldwide. It has now been reported in beef cattle and also sheep (contagious ovine digital dermatitis [CODD]). Three Treponema phylogroups are consistently isolated from lesions, Treponema medium- like, Treponema phagedenis -like, and Treponema pedis . The gastrointestinal (GI) tract and feces are suggested sites of treponemal infection in dairy cattle; however, isolation of DD-associated treponemes from these areas has previously failed. This study surveyed gingival tissues, rectal tissues, and feces of beef cattle and sheep for the molecular presence (PCR) and isolation of the three cultivable DD-treponeme phylogroups. Of the sheep gingival ( n = 40) and rectal ( n = 40) tissues, 1/40 gingival tissues was positive for DD-associated treponemes ( T. pedis ), as were 3/40 rectal tissues (one containing T. medium- like and two containing T. pedis ). No DD-associated treponeme DNA was amplified from beef cattle rectal tissues ( n = 40); however, 4/40 beef gingival tissues were positive for DD-associated treponemes (all containing T. phagedenis -like). A T. phagedenis -like DD-associated treponeme was isolated from the rectal tissue of a CODD symptomatic sheep. Beef cattle ( n = 41) and sheep ( n = 79) feces failed to amplify DD-associated Treponema DNA. Twenty-two treponemes were isolated from sheep feces; however, upon phylogenetic analysis, these clustered with the considered nonpathogenic treponemes. This study detected DD-associated treponemes in the GI tract tissues of sheep and beef cattle and successfully isolated a DD-associated treponeme from ruminant rectal tissue. This gives evidence that the GI tract is an important infection reservoir of DD-associated treponemes in multiple DD-infected species.