In situ imaging of molecular markers on a physical chromosome is an indispensable tool for refin-ing of genetic maps and validation genome assembly at the chromosomal level. Despite tremen-dous progress in genome sequencing the plant genome assembly at chromosome level still remain a challenge. Recently developed optical and Hi-C mapping aim to assist in genome assembly. For high-confidence in the genome assembly at chromosome level more independent approaches will be required. The present study aimed to refined an ultrasensitive Tyr-FISH technique and to de-velop a reliable and easy method for in situ mapping of a short unique DNA sequences on plant chromosomes. We have carefully analyzed the critical steps of the Tyr-FISH technique to find out the reasons for the failures of using the method. It has been shown that successful visualization of marker/gene depends significantly on method of chromosome slide preparation, probe design and labelling, high stringency washing. Appropriate adjustment of these steps allowed us to detect a short DNA sequence of 1.7Kb with a frequency of 51.6%. Based on our results, we developed a reliable and simple protocol for dual-color Tyr-FISH visualization of short unique DNA sequences on plant chromosomes. New protocol allows more accurate determination of the physical distance between markers and can be applied for faster integration of genetic and cytogenetic maps.