A separation algorithm that uses three wavelengths for the simultaneous measurement of the two-dimensional refractive index distribution and thickness profile of transparent samples is proposed. The optical system is based on a Mach-Zehnder interferometer with laser diode-based multiwavelength sources. A liquid crystal retarder is used to obtain interference images at four states with phases, and the optical phase of the object is then calculated with a four-bucket algorithm. A glass rod and several samples, including a slide glass, a glass wafer, and a cover glass are used to obtain experimental results at wavelengths of 635, 660, and 675 nm. The refractive indices of the sample are distributed with an accuracy of less than 0.0003 and the thickness profile is calculated on the basis of the measured refractive index. This result demonstrates that the proposed algorithm can be used to separate the refractive index distribution and thickness profile of samples in two dimensions.