Variation in pathogenicity (virulence and aggressiveness) of various single-oospore isolates of Sclerospora graminicola, the causal organism of downy mildew in pearl millet, was studied. Oospore isolates were maintained as sporangial progenies on the susceptible pearl millet genotype 7042-S through asexual generations. Twenty single-oospore isolates obtained from samples of oospore inocula from three locations (the ICRISAT Centre, Coimbatore, and Hisar) in India, were tested for pathogenicity on 7042-S. All the isolates were virulent on 7042-S and they varied considerably, between and within locations, in infection efficiency (11–44% infection). The S. graminicola populations from the ICRISAT Centre and Hisar were significantly more aggressive than those from Coimbatore. Considerable variation was found among the 10 single-oospore isolates from the ICRISAT Centre in infection efficiency and for latent period on a set of resistant and susceptible genotypes. Seven representative isolates, two each from the ICRISAT Centre (ICOS) and Coimbatore (CBOS), and three from Hisar (HSOS), exhibited variation in infection efficiency, sporangial production capacity, and oospore production rating on the susceptible genotypes 7042-S and NHB 3. A pathogenic fitness index (PFI) was calculated for these isolates as the product of the reciprocal of the latent period × percentage infected seedlings × log10 sporangia per cm2 leaf area × oospore production rating. Aggressiveness of these isolates, measured as PFI, varied greatly. CBOS-1 was the least aggressive with a PFI of 8·5 on NHB 3 and 69·6 on 7042-S; the PFI of the other isolates ranged from 81·1 to 370·5. Downy mildew resistance in three of the five known resistant lines (700651, P7-4 and 7042-R) was consistently effective against all 10 ICOS isolates, indicating that the resistance of these three lines is likely to be more stable, at least at the ICRISAT location, than that of the other two resistant lines.