Quantification of 11 clinical strains of Mycobacterium avium subsp. paratuberculosis isolated from domestic (cattle, sheep, and goat) and wildlife (fallow deer, deer, wild boar, and bison) animal species in an automatic liquid culture system (Bactec MGIT 960) was accomplished. The strains were previously isolated and typed using IS1311 PCR followed by restriction endonuclease analysis (PCR-REA) into type C, S, or B. A strain-specific quantification curve was generated for each M. avium subsp. paratuberculosis strain by relating the time to detection in the liquid culture system to the estimated log(10) CFU in each inoculum. According to their growth curves, the tested M. avium subsp. paratuberculosis strains were classified into two distinct groups. The first group included the S-type strain isolated from goat and all the sheep strains with C, S, and B genotypes. A second group contained the C- and B-type strains isolated from cattle, goat, and wildlife animals with the exception of the fallow deer strain. The strains isolated from cattle or sheep showed similar strain-specific standard curves irrespective of their genotype. In contrast, the strains isolated from goat or from wildlife animal species varied in their rates of growth in liquid culture. Universal-standard curves and algorithms for the quantification of each group of strains were generated. In addition, the liquid culture system was compared with a real-time quantitative PCR system for the quantification of the 11 M. avium subsp. paratuberculosis strains. Correlations between the estimated log(10) CFU and M. avium subsp. paratuberculosis DNA copy numbers were very high for all the tested strains (R ≥ 0.9).