Using a new technique to improve the sensitivity to weak quasi-periodic oscillations (QPOs), we discovered a new QPO peak at about 1100 Hz in the March 1996 outburst observations of 4U 1608-52, simultaneous with the similar to 600-900 Hz peak previously reported from these data. The frequency separation between the upper and the lower QPO peaks varied significantly from 232.7 +/- 11.5 Hz on March 3 to 293.1 +/- 6.6 Hz on March 6. This is the first case of a variable kilohertz peak separation in an atoll source. We discuss to what extent this result could be accommodated in beat-frequency models such as proposed for the kilohertz QPOs. We measured the rms fractional amplitude of both QPOs as a function of energy, and we found that the relation is steeper for the lower than for the upper frequency peak. This is the first source where such a difference between the energy spectrum of the two kilohertz QPOs could be measured.