Chromatic aberration had long been underestimated in conventional multicolor fluorescence microscopy of biological samples. Emerging new technologies with increasing spatial resolution, penetration depth has prompted the need for more accurate methods for chromatic correction. We have developed a robust, marker-free approach for highly accurate 3D measurement and correction of chromatic shifts in conventional and super-resolution 3D multicolor fluorescence microscopy datasets. We demonstrate that our method is able to correct the shifts to a 3D accuracy of ~10–20 nm depending on the method. After shift correction, distances between two objects could be measured with precision far beyond the classical resolution of light microscopes.