The sampling frequency offset (SFO) will induce phase rotation, inter-symbol interference and inter-carrier interference, which may seriously deteriorate the receiver’s performance in asynchronous optical discrete multi-tone (DMT) systems. In this work, a high-accuracy training symbols-based SFO estimation (SFOE) method is studied in both the additive white Gaussian noise channel and short-reach directly-modulated laser-based DMT transmission system. Two general SFO compensation (SFOC) methods, i.e., digital interpolation (DI) and phase compensation (PC), are comparatively investigated through both simulations and experiments. The results indicate that the accuracy of the SFOE method can be within ±0.2 ppm under the SFO range of ±500 ppm and different received optical powers. With the high-accuracy SFOE method, the 4th order DI has the best compensation performance compared to PC, 2nd and 3rd order DI methods. Moreover, the negligible EVM performance penalty compared with the case of SFO-free can also be achieved in an optical DMT transmission system. Besides, the PC-based SFOC method has similar EVM performance to the 4th order DI in the case of small SFO within ±300 ppm. •A high-accuracy training symbols-based SFO estimation method is proposed for short-reach DMT transmission.•The accuracy of the proposed SFOE is within ±0.2 ppm under the SFO range of ±500 ppm and different received optical powers.•Two commonly used SFO compensation methods of phase compensation and digital interpolation are experimentally investigated.