The longest possible haplotype is chromosome haplotype that is a set of co-inherited alleles occurred on a single strand chromosome inherited from one parent. Standard whole-genome shotgun sequencing technologies are limited by the inability to independently study the haplotype of homologous chromosomes due to the short-reads sequencing strategy and disturbance of homologue chromosomes. Here, we investigated several types of chromosomal abnormalities by a dilution-based method to separate an intact copy of homologous chromosome from human metaphase cells, and then single chromosomes were independently amplified by whole-genome amplification methods, converted into barcoded sequencing libraries, and sequenced in multiplexed pools by Illumina sequencers. We analyzed single chromosome derived from single metaphase cells of one patient with balanced chromosomal translocation t(3;5)(q24;q13), one patient with (47, XXY) karyotype and one with (47, XY, 21+) Down syndrome. We determined the translocation region of chromosomes in patient with t(3;5)(q24;q13) balanced chromosomal translocation by shallow whole-genome sequencing, which is helpful to pinpoint the chromosomal break point. We showed that SCS can physically separate and independently sequence three copies of chromosome 21 of Down syndrome patient. SCS has potential applications in personal genomics, single-cell genomics, and clinical diagnosis, particularly in revealing chromosomal level of genetic diseases.