The juncture between the west–east and north–south contrasting Holocene climatic domains across the Mediterranean is complex and poorly understood. Diatom analysis of Lake Dojran (Republic of Macedonia/Greece) provides a new insight into lake levels and trophic status during the Lateglacial and Holocene periods in the northeastern Mediterranean. Following a very shallow or even desiccated state at the core base at ca. 12,500 cal yr BP, indicated by sedimentological and hydro-acoustic data, diatoms indicate lake infilling, from a shallow state with abundant benthos to a plankton-dominated relatively high lake level and eutrophic state thereafter. Diatom-inferred shallowing between ca. 12,400–12,000 cal yr BP and a very low lake level and eutrophic, oligosaline state between ca. 12,000–11,500 cal yr BP provide strong evidence for Younger Dryas aridity. The earliest Holocene (ca. 11,500–10,700 cal yr BP) was characterised by a high lake level, followed by a lake-level reduction and increased trophic level between ca. 10,700–8,500 cal yr BP. The lake was relatively deep and exhibited peak Holocene trophic level between ca. 8,500–3,000 cal yr BP, becoming shallow thereafter. The diatom data provide more robust evidence and strengthen previous lake-level interpretation based on sedimentological and geochemical data during the earliest, mid and late Holocene, and also clarify previous uncertainty in interpretation of Lateglacial and early-Holocene lake-level change. Our results are also important in disentangling regional climate effects from local catchment dynamics during the Holocene, and to this end we exploit extant regional palynological evidence for vegetation change in the highlands and lowlands. The importance of seasonality in driving Holocene climate change is assessed by reference to the summer and winter latitudinal temperature gradient (LTG) model of Davis and Brewer (2009). We suggest that increased precipitation drove the high lake level during the earliest Holocene. The early-Holocene low lake level and relatively high trophic state may result climatically from high seasonality of precipitation and locally from limited, nutrient-rich catchment runoff. We argue that the mid-Holocene relatively deep and eutrophic state was driven mainly by local vegetation succession and associated changes in catchment processes, rather than showing a close relationship to climate change. The late-Holocene shallow state may have been influenced by a temperature-induced increase in evaporative concentration, but was coupled with clear evidence for intensified human impact. This study improves understanding of Lateglacial and Holocene climate change in the northeastern Mediterranean, suggests the important role of the LTG on moisture availability during the Holocene, and clarifies the influence of catchment processes on palaeohydrology.