The presence of tRNA genes in bacteriophages has been explained on the basis of codon usage (tRNA genes are retained in the phage genome if they correspond to codons more common in the phage than in its host) or amino acid usage (independent of codon, the amino acid corresponding to the retained tRNA gene is more common in the phage genome than in the bacterial host). The existence of a large database of sequenced mycobacteriophages, isolated on the common host Mycobacterium smegmatis, allows us to test the above hypotheses as well as explore other hypotheses for the presence of tRNA genes. Our analyses suggest that amino acid rather than codon usage better explains the presence of tRNA genes in mycobacteriophages. However, closely related phages that differ in the presence of tRNA genes in their genomes are capable of lysing the common bacterial host and do not differ in codon or amino acid usage. This suggests that the benefits of having tRNA genes may be associated with either growth in the host or the ability to infect more hosts (i.e., host range) rather than simply infecting a particular host.