Fluorescence in-situ hybridization (FISH) is a quick and affordable approach to map DNA sequences to specific chromosomal regions. Although FISH is one of the most important physical mapping techniques, research on the resolution of FISH on different cytological targets is scarce in plants. In this study, we report the resolution of FISH mapping on mitotic prometaphase chromosomes, meiotic pachytene chromosomes and extended DNA fibers in rice. A majority of the FISH signals derived from bacterial artificial chromosome (BAC) clones separated by approximately 1 Mb of DNA cannot be resolved on mitotic prometaphase chromosomes. In contrast, the relative positions of closely linked or even partially overlapping BAC clones can be resolved on a euchromatic region of rice chromosome 10 at the early pachytene stage. The resolution of pachytene FISH is dependent on early or late pachytene stages and also on the location of the DNA probes in the euchromatic or heterochromatic regions. We calibrated the fiber-FISH technique in rice using seven sequenced BAC clones. The average DNA extension was 3.21 kb/μm among the seven BAC clones. Fiber-FISH results derived from a BAC contig that spanned 1 Mb DNA matched remarkably to the sequencing data, demonstrating the high resolution of this technique in cytological mapping.