The colour of a horse's coat has long been discussed to reflect its temperament. One opinion is that Silver coloured horses are nervous, difficult to handle and react more strongly to different fear stimuli. The objective with the present study was to investigate if mutations associated with the Silver coat colour affect fear reactions in Icelandic horses. The hypothesis was that horses with the Silver mutation Arg618Cys in PMEL show stronger fear reactions than horses without the mutation (e.g. Black/Brown or Chestnut horses). Twenty-seven Icelandic horses (nine Silver, nine Chestnuts and nine Black/Brown) matched for sire, were exposed to the fear stimulus (a suddenly moving plastic bag) while feeding from a container. The test was repeated five times and behavioural responses and latency to return to the feed container were recorded. All horses were genotyped for the Silver mutation. The proportion of Silver horses that were hesitant to approach the test set-up before each trial was significantly higher (P < 0.05) than the proportion of non-Silver horses and this difference was most pronounced before the first two trials. No differences in reaction vigour or latency to resume feeding were found between the differently coloured horses. The results suggest that Silver horses are more cautious in novel situations rather than more reactive in fearful situations. One likely explanation for this difference is that the Silver mutation is associated with multiple congenital ocular anomalies (MCOA) syndrome and visual impairment. Furthermore, offspring (regardless of coat colour) from sires with a Best Linear Unbiased Prediction (BLUP, an index indicating which traits a horse will pass on to its offspring) value above 100 for the temperament trait ‘Spirit’, showed a greater fear reaction (P < 0.01) and reacted for a longer time (P < 0.01) than horses from sires with a lower (<100) index. These results indicate that horses with a high BLUP value for ‘Spirit’ seem to express stronger fear reactions. Breeding for Silver coat colour and the ‘Spirit’ trait, as it is currently defined, may need to be reconsidered if these results are confirmed in a larger cohort.