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Erwinia amylovoracan rapidly become highly resistant to applications of the antibiotic streptomycin through a single nucleotide mutation in the rpsLgene, which causes an amino acid substitution that prevents inhibitory binding of streptomycin while preserving the functionality of the ribosome. Several viable mutations are described in the literature, but screenings in the orchards consistently revealed that the one leading to the substitution of lysin by arginine at position 43 (K43R) is by far the most abundant. In this work, we tested the rate of emergence of the various spontaneous mutants, their tolerance to different antibiotic concentrations and their general fitness in competition experiments. Furthermore, we developed a molecular assay based on the SNaPshot™ Multiplex Kit from Thermo Fisher Scientific that allows swift determination of rpsLmutations using a single-base primer extension approach directly from colonies on plate. Or results indicate that the prevalence of variant K43R is largely due to fact that it retains the highest environmental fitness even in absence of antibiotic pressure. Sequence analysis of two resistant colonies that did not respond as expected in the SNaPshot™ assay revealed the existence of two yet undescribed rpsLmutations resulting in conditional-lethal streptomycin-dependent (SmD) phenotypes, which are unable to grow in absence of the antibiotic.