This article is the second in a 2-part series assessing the accumulated state of the transboundary Yukon River (YR) basin in northern Canada and the United States. The determination of accumulated state based on available long-term (LT) discharge and water quality data is the first step in watershed cumulative effect assessment in the absence of sufficient biological monitoring data. Long-term trends in water quantity and quality were determined and a benchmark against which to measure change was defined for 5 major reaches along the YR for nitrate, total and dissolved organic carbon (TOC and DOC, respectively), total phosphate (TP), orthophosphate, pH, and specific conductivity. Deviations from the reference condition were identified as “hot moments” in time, nested within a reach. Significant increasing LT trends in discharge were found on the Canadian portion of the YR. There were significant LT decreases in nitrate, TOC, and TP at the Headwater reach, and significant increases in nitrate and specific conductivity at the Lower reach. Deviations from reference condition were found in all water quality variables but most notably during the ice-free period of the YR (May–Sept) and in the Lower reach. The greatest magnitudes of outliers were found during the spring freshet. This study also incorporated traditional ecological knowledge (TEK) into its assessment of accumulated state. In the summer of 2007 the YR Inter Tribal Watershed Council organized a team of people to paddle down the length of the YR as part of a “Healing Journey,” where both Western Science and TEK paradigms were used. Water quality data were continuously collected and stories were shared between the team and communities along the YR. Healing Journey data were compared to the LT reference conditions and showed the summer of 2007 was abnormal compared to the LT water quality. This study showed the importance of establishing a reference condition by reach and season for key indicators of water health to measure change, and the importance of placing synoptic surveys into context of LT accumulated state assessments. Integr Environ Assess Manag 2013;9:439–455. © 2013 SETAC