Hesitation and alarm reactions of horses to visual features on the ground can be problematic in training and management. The reaction of riding horses to eight different coloured mats presented either on the ground or on the wall in an alleyway was recorded in order to assess the effect of the visual feature, colour, on horse behaviour. Horses had to walk over mats on the ground and walk past mats on the wall. Each colour and position combination was presented two times so that the effect of previous experience could be examined. The time taken to walk through the alleyway and the horses’ reactions to the colour were both related to the position of the colour and whether the horse had previous experience with that colour/position combination. The highest percentage of adverse reactions was observed during the initial presentation of colours on the ground. The colours yellow, white, black and blue, when encountered for the first time on the ground, caused more adverse reactions and an increase in time taken to walk through the alleyway than the colours green, red, brown and grey. During the second presentation of coloured mats on the ground, behavioural differences in response to colour were observed, but no difference in the time taken to walk through the alleyway was noted. No effects of colour were found when the mats were presented against the wall. The results of this study indicate that the colour of flooring can be controlled in order to minimize adverse behavioural reactions of horses, particularly during initial training.