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Several studies have reported Miocene shallow-marine incursions in the Pebas Megawetland system (Western Amazonia) based on paleontological and sedimentological evidence. Only a few publications restricted to Middle and Late Miocene fossils outcropping in the Iquitos (Peru), Benjamin Constant and Eirunepé (Brazil) areas have provided isotopic evidence to test these incursions. In this study, we present new carbon, oxygen, and strontium (87Sr/86Sr ratios) isotopic data of macrofossils (pacu and ray teeth, crab claws and oysters), and microfossils (ostracods and foraminifera) from reported lower to lower upper Miocene sedimentary rocks of Western and Eastern Amazonian (Peru and Brazil), mainly from shallow-marine deposits. It was also analyzed present-day organisms from distinct environments (beach, sea, riverine, and estuary) and compared these results with Miocene fossils. While C and O isotopic compositions indicate that Western Amazonia fossils display an isotopic composition similar to the present-day continental, limnic and riverine, environments (−17‰ to −5.61‰ for δ13C and − 10.14‰ to −1.92‰ for δ18O), Eastern Amazonia fossils display C and O isotopic compositions comparable to those from seawater environments (−2.53‰ to −0.72‰ for δ13C and − 4.85‰ to −1.76‰ for δ18O). We further used the 87Sr/86Sr isotopic composition of the fossils to estimate paleosalinity conditions. The 87Sr/86Sr isotopic compositions indicates that Eastern Amazonian water bodies of the Pirabas Formation (Marajó Basin) varied from mesohaline to marine conditions. In contrast, Early Miocene Western Amazonian water bodies of the Pebas Megawetland System (PMWS) are characterized by freshwater conditions. Nonetheless, in Middle and Early Late Miocene, oligohaline conditions are found in the Peruvian part of the PMWS. Paleosalinity estimates of the Brazilian part of the PMWS are less constrained and might indicate a salinity range between freshwater or oligohaline conditions. Our isotopic data suggest the PMWS was occasionally submitted to oligohaline conditions during Middle and Early Late Miocene shallow-marine incursions. We suggest that Miocene shallow-marine incursions in Western Amazonia at this time were characterized by transitional paleoenvironments, reconciling isotopic, sedimentological and paleontological evidences. • 374 Miocene Amazonian fossils attributed to shallow marine intervals analyzed. • Isotopic analyses of lower Miocene fossils from Eastern Amazonia indicate mesohaline to seawater conditions. • Isotopic data of middle and lower upper Miocene fossils from Peru yields oligohaline conditions in the Pebas Megawetland. • Marine incursions in Western Amazonia studied here occurred in transitional paleoenvironments with low seawater contribution. [ABSTRACT FROM AUTHOR]