Micro-millisecond range electric field pulses have been used for decades to facilitate DNA transfer into cells and tissues, while the growing number of clinical trials underline the strong potential of DNA electroporation. In this work, we present new sub-microsecond range protocols and methodology enabling successful electrotransfection in the sub-microsecond range. To facilitate DNA transfer, a 3 kV/60 A and high frequency (1 MHz) sub-microsecond range square wave generator was applied in the study. As a model, Chinese hamster ovary (CHO-K1) cells were used. Sub-microsecond range (300–700 ns) high frequency pulsed electric fields of 2–15 kV/cm were applied. The efficiency of electrotransfection was evaluated using two green fluorescent protein encoding plasmids of different size (3.5 kbp and 4.7 kbp). It was shown that transfection efficiency cannot be effectively improved with increase of the number of pulses after a certain threshold, however, independently on the plasmid size, the proposed sub-microsecond range pulsing methodology (2–5 kV/cm; n = 250) efficiency-wise was equivalent to 1.5 kV/cm × 100 μs × 4 electroporation procedure. The results of the study are useful for further development of in vitro and in vivo methods for effective electrotransfer of DNA using shorter pulses.