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Objective—To describe an epidemiological investigation of a bovine tuberculosis outbreak on a Colorado dairy operation. Procedures—A cull dairy cow infected with Mycobacterium bovis (index cow) was detected at a Texas abattoir during routine slaughter surveillance and subsequent diagnostic testing. This initiated an epidemiological investigation that was performed in accordance with USDA regulations. Results—The index cow was traced back to a Colorado dairy (index herd). Of the 908 cattle in the index herd, 101 (11.1%; 86 adult cattle > 2 years old and 15 immature cattle ≤ 2 years old) were infected with M bovis. Fourteen M bovis–infected cattle ≤ 2 years old were identified on 5 additional premises that had purchased cattle from the index herd directly or indirectly. All 115 affected cattle were infected with the same genetic type (spoligotype) of M bovis. A substantial proportion of cattle that left the index herd during the 5 years previous to the identification of the index cow were untraceable because of a lack of unique animal identification and inadequate records. Conclusions and Clinical Relevance—Results indicated that neonatal calves can have an important role in the transmission of M bovis. Also, this report highlights the exigent need for unique individual identification of livestock, including neonatal animals, so that thorough epidemiological investigations of reportable (zoonotic or foreign animal) diseases can be conducted when necessary.