For the first time, sections of the Upper Miocene Ishim Formation in the south of Tyumen oblast near the villages of Pyatkovo, Masali and Bigila were thoroughly studied by the palynological method. A series of mineralogical analyses of these sediments and radiocarbon analysis of the Quaternary sediments overlying the Ishim Formation were carried out. Four palynological assemblages (PA) were established in the Ishim Formation: PA1 with Botryococcus; PA2 with Botryococcus and Sigmopollis; PA3 with Alnus, Polypodiaceae, Botryococcus, and Sigmopollis; PA4 with Betula, Alnus, and Corylus. The layers with PA1, PA2, and PA3 were observed in two sections near Masali and Bigila. PA4 was found in sands and aleuropelites in the outcrop near Pyatkovo; it contained a significant amount of diverse pollen of temperate thermophylic broad-leaved taxa and scarce elements typical of Miocene (Taxodiaceae, Nyssa, and Tsuga). PA3 and PA4 were compared to the known complexes of Neogene sediments in Western Siberia. PA5 with Betula, Herbae, and Fungi was discovered in the bedded silts overlying the Ishim Formation in Masali outcrop. Previously, these sediments were attributed to the Late Miocene Pavlodar Formation. The composition and the structure of PA5 suggested Quaternary age of the hosting sediments. Radiocarbon analysis of the organic substance occurring in the silts has shown that these sediments were accumulated in the Late Pleistocene (Sartan Ice Age). For the first time, the information about microphytoplankton (Botryococcus, Pediastrum, Zygnemataceae, Sigmopollis) and other non-pollen palynomorphs contained in the Ishim Formation (Upper Miocene) and in Pleistocene sediments is presented. Development stages of the Late Miocene Ishim Basin were distinguished, the vegetation surrounding this basin was described, and the depositional environment of Pleistocene sediments (Masali outcrop) was reconstructed using palynological data.