We report on a spectral splitting effect of the cavity-less lasing emission of nitrogen ions at 391.4 nm pumped by 800-nm femtosecond laser pulses. It was found that with the increase of the nitrogen gas pressure and pump pulse energy, both R and P branches experience spectral splitting. With an external injected seeding pulse, a similar split spectral line is observed for the amplified emission. In contrast, for the fluorescence radiation, no such spectral splitting phenomenon is observed with much more abundant R branch structures. Our theoretical model considers gas ionization by the pump pulse, the competition of excitation of all relevant electronic and vibrational states, and an amplification of the seeding pulse in the plasma with a population inversion. Our simulation reproduces this spectral splitting effect, which is attributed to the gain saturation resulting in the oscillation of the amplitude of the amplified signal.