Fosfomycin is a first line antibiotic indicated for the treatment of acute uncomplicated cystitis in adult women infected with Escherichia coli or Enterococcus faecalis. Resistance to fosfomycin can be conferred by plasmid-transferable fos genes, which encode modified versions of glutathione transferase enzymes that can inactivate this antibiotic. In Canada, fosfomycin resistance and fos gene detection among E. coli clinical isolates are rare and have not been well described to date. Here, we characterize the fosA genes from three E. coli clinical isolates recovered from Canadian patients (two from urine, one from blood), and two additional fosA genes from the fosA7.5 family. Phylogenetic analysis of these FosA sequences showed close homology to previously characterized FosA proteins. Cloning and overexpression of each fosA sequence in E. coli BW25113 for antimicrobial susceptibility testing confirmed that all fosA genes exhibited fosfomycin-resistant phenotypes (MIC values of >512 and >1024 µg/mL) with the exception of one fosA7.5 variant. Homology modelling of each FosA protein suggests that residue alterations in this inactive fosA7.5 variant may impact fosfomycin drug interactions at the active site region. The findings from this study identify and functionally characterize FosA3, FosA8, as well as novel FosA7.5 members and highlight the importance of phenotypic characterization of fosA genes.