Identifying the geochemical fingerprints of fluids that return to the surface after high volume hydraulic fracturing of unconventional oil and gas reservoirs has important applications for assessing hydrocarbon resource recovery, environmental impacts, and wastewater treatment and disposal. Here, we report for the first time, novel diagnostic elemental and isotopic signatures (B/Cl, Li/Cl, δ11B, and δ7Li) useful for characterizing hydraulic fracturing flowback fluids (HFFF) and distinguishing sources of HFFF in the environment. Data from 39 HFFFs and produced water samples show that B/Cl (>0.001), Li/Cl (>0.002), δ11B (25.31‰) and δ7Li (6.10‰) compositions of HFFF from the Marcellus and Fayetteville black shale formations were distinct in most cases from produced waters sampled from conventional oil and gas wells. We posit that boron isotope geochemistry can be used to quantify small fractions (~0.1%) of HFFF in contaminated fresh water and likely be applied universally to trace HFFF in other basins. The novel environmental application of this diagnostic isotopic tool is validated by examining the composition of effluent discharge from an oil and gas brine treatment facility in Pennsylvania and an accidental spill site in West Virginia. We hypothesize that the boron and lithium are mobilized from exchangeable sites on clay minerals in the shale formations during the hydraulic fracturing process, resulting in the relative enrichment of boron and lithium in HFFF.