As a great number of the genomic DNA sequences can now be generated in a day, the demand for a very fast and accurate method for positioning genomic DNA sequences on a genome is high. A unique marker is a sequence which appears only once in a genome. The unique marker method is an efficient method to perform this task. The amount of time needed for positioning genomic DNA sequences grows rapidly when we use longer unique markers. On the other hand, the success rate of short unique markers is not very high. In this paper, we propose a multi-layer genome-wide unique marker positioning (MUGUP) technology for obtaining a high rate of accuracy as well as a high speed of computation. Our method combines the benefits of both short and long unique markers. We also compared our method with two other famous methods: BLAST and SSAHA. Our method was found much faster than these two methods.