Purpose In order to increase the simplicity and accuracy of performing the time alignment on a positron emission tomography (PET) scanner, a new generation timing alignment probe has been developed. Methods A timing alignment probe containing a plastic scintillator with an embedded sodium-22 source which is optically coupled to a fast photomultiplier tube (PMT) is described and tested. When a positron is ejected from the radioactive atom’s nucleus, its kinetic energy is absorbed in and can be detected as a light flash from the scintillator. This is used as the reference time for each atom’s positron decay. It is only after the positron slows that it can combine with an electron, forming positronium after which the 511 keV annihilation photons will be created, possibly traveling to the PET detectors. In practice, the probe is placed in the center of the scanner’s field of view and connected to the coincidence circuit. Since the delay between an annihilation photon’s detection and positron detection is almost identical (the lifetime of positronium in a solid is extremely short, and the gamma rays’ path lengths are equal with the probe in the center of the scanner), the probe’s signal provides a fixed reference time to which the response of individual crystals in the PET detectors can be compared. We present an evaluation of the performance of this probe. We first investigated the intrinsic performance of the time-alignment probe comparing its timing resolution with two barium fluoride crystals in coincidence. We then investigated the timing performance of the probe in coincidence with various individual scintillation crystals and with detectors from two commercial PET scanners. Results The best full-width at half-maximum (FWHM) timing resolution of the probe was found when in coincidence with BaF 2 at 400 ps. The common commercial scintillator lutetium oxy-orthosilicate (LSO) was tested and its FWHM was 510 ps. When testing the crystal arrays used in two commercial block detectors it was found that there are significant, systematic timing delays among the crystals. In the Siemens HiRez ® detector the average time difference between the positron detection and annihilation photon detection is 500 ps with a standard deviation of 115 ps. In the Siemens Focus ® detector the average was 1000 ps and a standard deviation of 400 ps. The reasons for the variation in apparent arrival times among the crystals appear to be due to the electronic readout rather than light transport from the crystals to the PMT. Conclusion Many PET scanners use a global time offset for each detector, and these variations in apparent time delay from individual crystals in the detector would have a significant impact if these detectors were employed in a time-of-flight (ToF) PET scanner.