The aim of this study was to demonstrate, with molecular techniques, that Dactylis glomerata (orchard grass) is an alternate host of yellow rust of barley (Hordeum vulgare); by monopustular culture of the pathogen, microscopy and molecular analysis. Puccinia striiformis causes the disease called yellow rust in annual crop plants, mainly barley (Hordeum vulgare), also wheat (Triticum aestivum), oats (Avena sativa), rye (Secale cereale) and is found in perennial pastures such as the orchard grass, reducing the palatability of the pastures, harming animal nutrition (sheep and cattle). Due to these characteristics, orchard grass was proposed as a possible alternate host for yellow rust of barley. In order to standardize the monospustular culture methodology of P. striiformis, an experimental trial was carried out evaluating adhesives (A) and times (T) in a cold-dark chamber using the completely randomized design (CRD) with a 3 A x 3 T factorial arrangement with four repetitions. Using the uredospores obtained from the monopustular culture of P. striiformis, microscopic measurements were performed, DNA was extracted, PCR and sequence alignment were performed using the BLAST and CLUSTAL software for the ITS region. The results show that the uredospores of yellow rust of orchard grass and barley have similar size and color. The results of the analysis of the sequence alignment of the DNA sequences of the yellow rust ITS gene in the two host species presented high similarity at the molecular level (99%), demonstrating that orchard grass is an alternate host of barley yellow rust. These results should be taken into consideration for rotations and associations of grasses used for livestock feeding.