Effects-based analysis is a fundamental component of watershed cumulative effects assessment. This study conducted an effects-based analysis for the Peace-Athabasca-Slave River System, part of the massive Mackenzie River Basin, encompassing 20% of Canada's total land mass and influenced by cumulative contributions of the W.A.C. Bennett Dam (Peace River) and industrial activities including oil sands mining (Athabasca River). This study assessed seasonal changes in 1) Peace River water quality and quantity before and after dam development, 2) Athabasca River water quality and quantity before and after oil sands developments, 3) tributary inputs from the Peace and Athabasca Rivers to the Slave River, and 4) upstream to downstream differences in water quality in the Slave River. In addition, seasonal benchmarks were calculated for each river based on pre-perturbation post-perturbation data for future cumulative effects assessments. Winter discharge (January-March) from the Peace and Slave Rivers was significantly higher than before dam construction (pre-1967) ( p < 0.05), whereas summer peak flows (May-July) were significantly lower than before the dam showing that regulation has significantly altered seasonal flow regimes. During spring freshet and summer high flows, the Peace River strongly influenced the quality of the Slave River, as there were no significant differences in loadings of dissolved N, total P (TP), total organic C (TOC), total As, total Mn, total V, and turbidity and specific conductance between these rivers. In the Athabasca River, TP and specific conductance concentrations increased significantly since before oil sands developments (1967-2010), whereas dissolved N and sulfate have increased after the oil sands developments (1977-2010). Recently, the Athabasca River had significantly higher concentrations of dissolved N, TP, TOC, dissolved sulfate, specific conductance, and total Mn than either the Slave or the Peace Rivers during the winter months. The transboundary nature of the Peace, Athabasca, and Slave River basins has resulted in fragmented monitoring and reporting of the state of these rivers, and a more consistent monitoring framework is recommended. Integr Environ Assess Manag 2013;9:405-425. © 2012 SETAC