Karyotype analysis is an effective tool in the diagnosis of chromosomal diseases. Image processing technology enables the analysis and medical description of the number and morphological characteristics of chromosomes. Chromosome images are characterized by the high cohesion, overlapping and nesting, which represents a difficult problem in chromosome image segmentation at this stage. To effectively segment overlapping chromosomes, this paper proposes an improved overlapping chromosome segmentation algorithm based on contour and skeleton. Based on the preprocessed chromosome karyotype map, the algorithm applies the Canny operator for chromosome contour detection and edge information extraction to accurately locate the skeleton and complete the segmentation. We evaluated our algorithm on 200 chromosome images, and the results showed the segmentation accuracy of “+” and “T” shaped overlapping chromosomes to be 97.8% and 96.1%, respectively, while the accuracy of overlapping chromosome segmentation in the whole image was 98.5%. This shows the effectiveness of the algorithm.