The study of errors in gene translation has largely been confined to a small number of model organisms. We have examined all possible misreading errors at a defined codon in Mycobacterium smegmatis. Using a dual-luciferase gain of function reporter system that employs a mutated essential lysine in firefly luciferase, we accurately quantified mistranslation errors. Overall, accuracy of gene translation was comparable with Escherichia coli at <1/2000 errors/codon during exponential growth. Stationary phase was associated with a dramatic increase in misincorporation errors by Lys-  at a subset of three codons, each with a single base changed from the AAG lysine codon. The maximum error rate detected was 0.2% with codon AUG. Treatment with streptomycin increased misreading errors at several codons associated in particular with U·U, G·U and C·U codon·anti-codon mismatches, but oxidative stress did not change translational fidelity. Our study is the first comprehensive examination of misreading errors for a defined codon in mycobacteria.