The Carboniferous was a period of intense environmental perturbations, climate changes between greenhouse and icehouse, eustatic sea level change, and accumulation of organic carbon-rich sediments. At this time, the Tyler Formation was deposited in the midcontinent USA. A detailed palynological analysis of the Tyler Formation revealed a highly diverse assemblage of spores with minor pollen content, represented by 100 species belonging to 51 genera. Stratigraphically constrained spores and pollen grains were used to construct three interval zones of middle-late Chesterian (late Viséan-Serpukhovian) to early Morrowan (middle Bashkirian) ages. The stratigraphic position of the Mississippian-Pennsylvanian boundary was determined in the lower Tyler Formation based on the last appearance of typical forms of the late Chesterian, including Tripartites vetustus, Knoxisporites triradiatus, Knoxisporites stephanephorus, Densosporites diatretus, and Schopfipollenites acadiensis, compared to the first appearance of early Morrowan Crassispora kosankei, Cirratriradites saturnii, Radiizonates aligerens, and Raistrickia saetosa. Palynofacies analysis and statistical clustering of the Tyler Formation showed three palynofacies assemblages. PFA-1 showed moderate relative abundances of phytoclasts and AOM, suggesting deposition close to fluvio-deltaic and shallow marine environments, while PFA-2 exhibited high abundances of phytoclasts, mostly of opaque wood, reflecting deposition in active river-dominated delta plains. PFA-3 showed the highest abundances of AOM, suggesting deposition in a shallow marine environment. Organic petrography and geochemistry data indicate that the Tyler Formation is one of the best source rock intervals throughout the midcontinent USA. Based on organic matter richness, the Tyler Formation is subdivided into three groups. The first group has TOC contents higher than 10 wt% of kerogen Types III, mixed II/III, and II with excellent hydrocarbon generation potential. The second group has TOC content in the range of 2–10 wt% of kerogen Types III, mixed II/III, and II, and fair to excellent hydrocarbon generation potential. The third group shows organic matter richness with TOC content below 2 wt% with good organic matter richness and kerogen Types III to IV, and poor to fair hydrocarbon generation potential. The organic matter thermal maturity is evaluated based on Tmax and VRo% values, suggesting that all samples of Tyler Formation are in the early to late stages of the oil window. However, care should be considered when assessing a mature source rock because the kerogen typing and generation is based on present-day TOC, S2, and HI rather than their original values.