An image sensor may include crosstalk calibration pixels. Crosstalk calibration pixels may include exposed pixels and shielded pixels. Exposed pixels may be partially or completely surrounded by shielded pixels. Calibration pixels may be formed in a checkerboard pattern of alternating shielded and exposed pixels or a double checkerboard pattern of alternating pairs of shielded and exposed pixels. Exposed pixels may have apertures of various size in a shielding layer that shields the shielded pixels from light. Signals generated by exposed and shielded pixels may be used in assessing pixel optical and electrical crosstalk and indirectly deducing the spectral composition of incoming light for particular locations in a pixel array. Information about local crosstalk across the array may be used in coordinate dependent color correction matrices, white balance algorithms, luminance and chroma noise cancellation, edge sharpening, assessment of pixel implantation depth, and measuring a modulation transfer function.