This paper aims at assessing the reproductive segregation within the endemic Barbus (renamed Labeobarbus) species flock of Lake Tana (Ethiopia). Migration, followed by temporal and spatial reproductive segregation in the upstream tributaries of two inflowing rivers was studied systematically over the 1999 and 2000 spawning seasons. Physical events that may trigger lacustrine migration and characterise suitability of spawning grounds were analysed. Six species migrate 30-40 km upstream Gumara River during declining flow, just after the rainy season. Spawning occurs in the well-oxygenated gravel beds of four Gumara tributaries. Eight 'large barb' species were absent from the rivers, or found only incidentally, thus segregating them at a macro-spatial scale from the six river-spawners. The missing species spawn either in the lake or in other rivers not sampled. Long distance migration and species-specific spawning sites suggest that homing may have facilitated reproductive isolation and speciation. A fine-tuning between homing and gonad development is suggested since females reach spawning maturity only as they arrive at the spawning grounds. This study provides convincing evidence for reproductive segregation, and contributes in unravelling the evolution of this unique Labeobarbus species flock. Collective migration and riverine spawning of six Labeobarbus species makes them very vulnerable for overfishing at the spawning areas.