Tire and road wear particles (TRWPs) are generated from friction between tires and the road and contain polymer tread with pavement encrustations. Single particle analysis (SPA) of tire source contribution in environmental samples has been limited by interferences in common spectroscopic polymer techniques. This study extends a density separation and chemical mapping protocol for road simulator generated TRWPs toward the identification and characterization of individual TRWPs in more complex road dust, roaddust-spiked artificial sediment, tunnel dust, and environmental settling pond sediment samples. TRWPs were identified by a combination of physical (elongated/round shape with variable amounts of mineral encrustation) and elemental surface characteristics [colocalization of (S + Zn/Na) ± (Si, K, Mg, Ca, and Al)]. Organic surface markers (C 7 H 7 + ), overlapping FTIR spectra with tread reference material, and resistance to heat-induced deformation were selectively used to confirm particle identification. The TRWP size displayed an increasing average trend of 54, 158, and 267 μm by number (94, 224, and 506 μm by volume) in tunnel dust, road dust, and environmental sediment, respectively. TRWP size distributions within road dust 10× diluted with artificial sediment agreed with those of pure road dust. Our SPA methodologies determined the size distribution of TRWPs in environmental sample types with increasing sample complexity.