Spoligotyping is a typing tool used worldwide for epidemiological studies on Mycobacterium tuberculosis complex organisms; however it has received little attention regarding improvement of its discriminatory power (DP). In this study, we have evaluated a spoligotyping membrane prepared with 25 novel spacer sequences selected from a previous study [van der Zanden AG, Kremer K, Schouls LM. Improvement of differentiation and interpretability of spoligotyping for Mycobacterium tuberculosis complex isolates by introduction of new spacer oligonucleotides. J Clin Microbiol 2002;40:4628–39] on 308 M. bovis and 88 M. caprae Spanish isolates in comparison with the traditional spoligotyping membrane. The results obtained by combining the two membranes together revealed an improvement of 45 patterns instead of 31. The spacers used in the second membrane were able to distinguish 8 out of the 16 M. bovis types that had more than one isolate. Seven of these types were differentiated into two subtypes with the second-generation membrane, while spb-7, the most prevalent in Spain, was further differentiated into eight subtypes. This second-generation membrane also differentiates M. bovis from M. caprae. A set of 39 spacers (1, 2, 4–8, 10–15, 17–21, 23, 26–32, 37, 44–49, 51–54, 56 and 57) contain all the DP for both M. bovis and M. caprae isolates; and a set of 35 spacers (1, 2, 4–8, 10–15, 17–21, 26–32, 37, 44–48, 52–54 and 57) had all the DP for the M. bovis isolates. Our results show that the research on new spacers and the design of a new membrane may be useful for epidemiological studies of M. bovis and M. caprae isolates.