Occurrences of antibiotic‐resistant Escherichia coli in two springs of a karstic system (NW France) providing drinking water were determined to study the role of aquifers in the dissemination of the resistance genes. Water samples were collected during wet and dry periods and after a heavy rainfall event to investigate E. coli density, antibiotic resistance patterns, and occurrences of class 1, 2, and 3 integrons. By observing patterns of the resistant isolates (i.e. number and type of resistances) and their occurrences, we were able to define two resistant subpopulations, introduced in the aquifer via surface water: (1) R1‐2, characterized by one or two resistance(s), essentially to chloramphenicol and/or tetracycline (96.5%), was always found during the heavy rainfall event; (2) R3‐10, characterized by three or more resistances, mostly resistant to tetracycline (94.1%) and beta‐lactams (86%), was found transiently. Class 1 and 2 integrons were detected, mostly in the R3‐10 subpopulation for class 1 integrons. The characteristics of these two subpopulations strongly suggest that the contamination originates from pasture runoff for the R1‐2 subpopulation and from wastewater treatment plant effluents for the R3‐10 subpopulation. These two subpopulations of E. coli could be used as biological indicators to determine the origin of groundwater contamination.

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