A total of 87 Mycobacterium bovis isolates from 475 intradermal tuberculin test (ITT)-positive cows during 2008 to 2011, and 39 M. bovis isolates from 77 ITT-positive deer from 2009 to 2010, were collected in the present study. The isolation rate of M. bovis in deer was significantly higher (50.6%, 39/77) than in cow (18.3%, 87/475). During 1997-2007, 24 isolates have been obtained without known from how many ITT positive cows. The distribution of 111 M. bovis isolates collected from cow during 1997 to 2011 revealed that 75 isolates in Pingtung County (76.5%), 18 in Yunlin County (18.4%), 11 in Chiayi County (11.2%), 3 in Taitung County (2.7%）, 1 in Taoyuan, Taichung, Changhua, and Tainan County (0.9%), respectively. Four patterns were identified among the 111 bovine isolates using a spacer oligonucleotide typing (spoligotyping) method compared with four patterns observed in the M. bovis molecular typing database (http://www.mbovis.org
). Pattern SB0265 was the dominant spoligotype (72.1% of the isolates), followed by SB0140 (25.2%), SB1182 (0.9%), and SB1040 (1.8%) respectively. Only two patterns of spoligotyping were identified from deer isolates (SB0265, 97.4% and SB0140, 2.6%, respectively). In general, this study demonstrated the isolation rate of M. bovis in deer was significantly higher than in cow (P<0.05), and SB0265 was the most common spoligotyping pattern isolates from both cow and deer in Taiwan.