Three hypervirulent strains of Mycobacterium tuberculosis isolated from patients suffering from tuberculous meningitis were shown to grow more rapidly inside human macrophages in our previous study. In the current investigation, genomic polymorphisms in these hypervirulent strains were examined using microarray-based comparative genomic hybridization. Among the five genomic polymorphisms identified, two are in-frame deletion (Rv0071/4 and Rv0613c/6c), two are frameshift deletion (Rv1758' and Rv2820c'), and one is gene replacement (Mb3159). The five genomic polymorphisms were transformed into Mycobacterium smegmatis strain mc(2)155 and the survivability of recombinants inside the human monocytic cell line THP-1 was measured. Interestingly, only the recombinant possessing the Rv2820c' survived significantly better than the vector control after 6 h of ex vivo infection (P < 0.001, one-way ANOVA). The Rv2820c' was later transformed into Mycobacterium marinum strain M and the recombinant was used to infect zebrafish. The in vivo infection also showed that the zebrafish infected with the recombinant possessing the Rv2820c' died significantly faster than the vector control (P = 0.006, log-rank test). The 3' truncation in the Rv2820c' was caused by the Beijing/W-defining deletion RD207 and is commonly found in the Beijing/W strains. The current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence ex vivo and in vivo. This enhancement, however, was not observed for the intact Rv2820c of the non-Beijing/W strains. The presence of the 3' truncated portion of Rv2820c may interfere with overall protein folding and render the Rv2820c of the non-Beijing/W strains non-functional.