Insulator-based dielectrophoresis can be used to manipulate biological particles, but has thus far found limited practical applications due to low sensitivity. We present linear sweep three-dimensional insulator-based dielectrophoresis as a considerably more sensitive approach for strain-level discrimination bacteria. In this work, linear sweep three-dimensional insulator-based dielectrophoresis was performed on Pseudomonas aeruginosa PA14 along with six isogenic mutants as well as Streptococcus mitis SF100 and PS344. Strain-level discrimination was achieved between these clinically important pathogens with applied electric fields below 10 V/mm. This low voltage, high sensitivity technique has potential applications in clinical diagnostics as well as microbial physiology research.National Science Foundation (U.S.) (Award 1150615)MISTI Hayashi Seed Fund