A method for the detection of diazepam in horse hair samples by low resolution gas chromatography-mass spectrometry (GC-MS) was developed. Two other techniques, gas chromatography-high-resolution mass spectrometry (GC-HRMS) and high-performance liquid chromatography-atmospheric pressure chemical-ionisation mass spectrometry (HPLC-APCI-MS-MS) were applied on some selected samples. Sample preparation was performed according to a technique previously described for human hair, involving incubation with Sorensen buffer and solvent extraction. Hair samples from different sites such as coat on the neck, coat on the back, mane and tail were collected from two thoroughbreds which had received several dosages of diazepam corresponding to a total dose of 750 mg and 200 mg of diazepam respectively. In the first experiment, by low resolution GC-MS using single ion monitoring, diazepam was detected in the mane for at least 85 d after the last administration. In the second one, using the same method, diazepam was detected in the coat on the neck up to 25 d following the last administration. Low resolution GC-MS data were confirmed by the two other techniques. Furthermore, GC-HRMS even made possible the detection of diazepam up to 38 d after the administration of 200 mg of diazepam.