During the last decade, isotopic fractionation has gained acceptance as an indicator of microbiological and chemical transformations of contaminants in groundwater. These transformation processes typically favor isotopically light, compared to isotopically heavy, contaminants, resulting in enrichment of the latter in the residual aqueous phase. In these isotope applications, it has been generally presumed that physical transport processes in groundwater have a negligible effect on isotopic enrichment. It is well known, however, that aqueous phase diffusion generally proceeds faster for isotopically light, compared to isotopically heavy, solute molecules, often resulting in isotopic fractionation in groundwater. This paper considers the potential for isotopic fractionation during transport in groundwater resulting from minute isotopic effects on aqueous diffusion coefficients. Analyses of transport in heterogeneous systems delimit the viable range of isotopic fractionation by diffusion in groundwater. Results show that diffusion can result in similar degrees of depletion and enrichment of isotopically heavy solutes during transport in heterogeneous systems with significant diffusion rate–limited mass transfer between fast- and slow-flow zones. Additional analyses and examples explore conditions that attenuate the development of significant fractionation. Examples are presented for ¹³C methyl tertiary butyl ether and deuterated and nondeuterated isopropanol and tertiary butyl alcohol using aqueous diffusion coefficients measured by the Taylor dispersion method with refractive index profiling as a part of this study. Examples elucidate the potential for diffusive fractionation as a confounder in isotope applications and emphasize the importance of hydrogeologic analysis for assessing the role of diffusive fractionation in isotope applications at contaminant field sites.