SummaryThree inoculation techniques for Karnal bunt (Tilletia indica) disease of wheat were compared: 1) boot inoculation ‐ injection of inoculum with a hypodermic syringe into the boot; 2) spray inoculation ‐ inoculum sprayed at growth stages between heading and anthesis, and 3) cotton wool inoculation ‐ small pieces of cotton wool saturated in inoculum placed either inside the floret or between the spikelet and rachis. Each inoculation technique was assessed using susceptible cultivars to determine the optimum inoculum concentration, the ideal plant growth stage and the humidity requirements for successful infection.Boot inoculation did not require high humidity and gave reliable infection with low secondary sporidia concentrations (1000–10 000/ml). The ideal plant growth stages for inoculation were early‐boot and mid‐boot. Spray inoculation required high secondary sporidia concentrations (50 000/ml) and 48 h of high humidity, but infection was initiated over a range of growth stages throughout heading and anthesis. Cotton wool inoculation gave low levels of infection at growth stages throughout heading and anthesis, even with high secondary sporidia concentrations (