Fosfomycin has attracted renewed interest for the treatment of lower urinary tract and even systemic infections caused by Gram-negative pathogens with resistance to traditionally used agents. The main concern regarding the clinical utility of fosfomycin refers to the potential for the emergence of resistance during therapy. In this review, we evaluate the available published evidence regarding the mechanisms and the frequency of in vitro mutational resistance to fosfomycin in Gram-negative pathogens. We also review data regarding the emergence of resistance in clinical studies of fosfomycin therapy in various infectious syndromes and data from studies that evaluate the evolution of fosfomycin resistance over time. There appears to be discordance between the high frequency of mutational resistance to fosfomycin in vitro and the lower extent of this phenomenon in clinical studies. This discordance could at least partly be attributed to a biological cost associated with common mutations that confer resistance to fosfomycin, including decreased growth rate and low adherence to epithelial cells for the resistant mutants. The development of resistance appears to be more frequent both in vitro and in clinical studies for Pseudomonas aeruginosa in comparison with Escherichia coli, whereas relevant data for other Enterobacteriaceae are relatively scarce. The urinary tract seems to provide a favourable environment for the use of fosfomycin with a low associated likelihood for the emergence of resistance, owing to high drug concentrations and acidic pH. Additional data are needed to further clarify the optimal use of fosfomycin for different infectious syndromes caused by contemporary multidrug-resistant pathogens.