Bovine tuberculosis (bTB) remains a problem on many dairy farms in Mexico, as well as a public health risk. We previously found a high frequency of Mycobacterium bovis DNA in colostrum from dairy cows using a nested PCR to detect mpb70. Since there are no reliable in vivo tests to determine the effectiveness of booster Mycobacterium bovis BCG vaccination against bTB, in this work we monitored M. bovis DNA in colostrum by using this nested PCR. In order to decrease the risk of adverse reactions in animals likely containing viable M. bovis, a single application of BCG and a subunit vaccine (EEP-1) formulated with M. bovis culture filtrate proteins (CFP) and a copolymer as the adjuvant was performed in tuberculin skin test-negative cattle (TST(-)), while TST reactor animals (TST(+)) received EEP-1 only. Booster immunization using EEP-1 was applied to both groups, 2 months after primary vaccination to whole herds and 12 months later to lactating cows. Colostrum samples were collected from 6 farms where the cows were vaccinated over a 12-month period postvaccination and, for comparison, from one control farm where the cows were not vaccinated with comparable bTB prevalence. We observed an inverse relationship between the frequency of M. bovis DNA detection and time postvaccination at the first (P < 0.001) and second (P < 0.0001) 6-month periods. Additionally, the concentration of gamma interferon (IFN-γ) was higher in mpb70 PCR-positive colostrum samples (P = 0.0003). These results suggest that M. bovis DNA frequency in colostrum could be a potentially useful biomarker for bTB vaccine efficacy on commercial dairy farms.