Tetrahymena thermophila is a eukaryotic model organism that contains two types of nuclei; the macronucleus (MAC) is the expressed nucleus and its genome has been sequenced, while the micronucleus (MIC) is the germline genome and is transcriptionally silent. During sexual reorganization: new macronuclei differentiate from mitotic products of the fertilization nucleus and the five micronuclear chromosomes are fragmented into 181 MAC chromosomes. Fragmentation occurs at a specific 15-bp sequence known as the chromosome breakage sequence (CBS); these sites reveal the relationship between the MIC and MAC genomes. The objective of this research is to use CBS to link MAC chromosome sequence scaffolds in their correct order and orientation on the left arm of MIC chromosome 4. We designed PCR primers at the end of each MAC chromosome scaffold, and then used Polymerase Chain Reaction to amplify the micronuclear region between the ends of two adjacent MAC chromosomes. Each 4L MAC chromosome end was tested in pair-wise combination with every other 4L MAC chromosome end. Specific PCR products were cloned and sequenced to confirm that their terminal nucleotide sequence was identical to that at the two chromosome ends with the previously unknown CBS sequence in between. Finding the location and orientation of the MAC chromosomes in the MIC will help in the MIC genome sequencing project which is currently underway.