A disk drive is disclosed comprising a disk comprising a track, wherein the track comprises a sync mark. The disk drive further comprises a head comprising a plurality of read sensors including a first read sensor, and a second read sensor separated from the first read sensor by a down-track spacing. A first time-stamp (TS1) is generated after detecting the sync mark using the first read sensor as the disk rotates, and a second time-stamp (TS2) is generated after detecting the sync mark using the second read sensor as the disk rotates. A calibration value representing the down-track spacing is generated based on the TS1 and the TS2.