The phylogenetic relationships for Ipomoea species are incongruent in previous studies. Comparative karyotype analysis can provide valuable information for phylogenetic relationships among species. A reliable and efficient system for chromosome identification is the foundation for karyotype analysis. However, for the Ipomoea species, individual chromosomes were not identified in all previous reported karyotypes due to their small size, high number and similar morphology. Fluorescence in situ hybridization (FISH) using oligonucleotides (oligos) as probes is a new strategy for chromosome identification and karyotype analysis. Here, we developed the first set of oligo-based probes based on the reference genome of Ipomoea nil, a model species in the genus Ipomoea. In all, we developed four oligo-FISH probes. By a combined use of four oligo probes, sequential FISH analysis were conducted on the same metaphase cells with each round including two probes, which permitted simultaneous identification of all I. nil chromosomes and anchoring 15 pseudochromosomes to individual cytological chromosomes. Moreover, 45S and 5S rDNA were mapped to specific chromosomes. A karyotype based on individually identified chromosomes was established, which was the first FISH-based molecular cytogenetic karyotype of I. nil. Our study has created the basis for studying chromosome variation and evolution in genus Ipomoea by comparative karyotype analysis using this set of oligo probes.