AbstractRiver organic matter transformations impact the cycling of energy, carbon, and nutrients. The delivery of distinct dissolved organic matter (DOM) sources can alter aquatic DOM cycling and associated biogeochemical processes. Yet DOM source and reactivity are not well‐defined for many river systems, including in western Canada. Here, we explore DOM cycling in the mainstem of the Oldman River (stream order 6–7), a heavily regulated river network in southern Alberta (Canada). We compared seasonal river DOM content, composition, and bioavailability with nine endmember leachates from the river valley using optical properties and incubations to estimate biodegradable dissolved organic carbon (BDOC). River DOM composition was most similar to terrestrial soil leachates, followed by autochthonous DOM leachates. River DOM bioavailability was low (BDOC = 0%–16.6%, mean of 7.1%). Endmember leachate bioavailability increased from soils (BDOC = 23.9%–53.7%), to autochthonous sources (fish excretion, macrophytes, biofilm; BDOC = 49.9%–80.0%), to terrestrial vegetation (leaves, shrubs, grass; BDOC > 80%), scaling positively with protein‐like DOM content and amount of leachable dissolved organic carbon (DOC), and negatively with aromaticity. Seasonally, DOC concentrations changed little despite >15‐fold increases in discharge during spring. River DOM composition shifted modestly toward soil‐like endmembers in spring and more bioavailable autochthonous end members in autumn and winter. Low DOM bioavailability in the river mainstem and low DOC yields shown in previous work point to limited internal processing of DOM and limited bioavailable DOM delivery to downstream habitats, possibly due to upstream flow regulation. Our observations provide important insights into the functioning of western Canadian aquatic networks.