Inverse PCR (IPCR) is a reliable, straightforward, and effective technique for acquiring unknown sequences. In this study, we used the model monocot Brachypodium distachyon (ecotype Bd21) to standardize the conditions and materials necessary for the successful execution of IPCR. The analysis of the amplified sequences resulted in the following conclusions. First, the distance between the nearest primer and the boundary of the known-unknown sequence is crucial for determining whether the target sequence can be expanded in the second round of IPCR. Specifically, this distance should exceed 100 bp, ideally around 200 bp. Second, because the random cleavage of a 6 bp endonuclease occurs at a greater distance than that of a 4 bp endonuclease, the use of a 6 bp endonuclease in IPCR results in larger but often inconsistent bands, while maintaining good specificity. Therefore, if the goal is to amplify longer sequences or achieve high accuracy, it is advisable to select endonucleases with 6 bp restriction sites. Third, IPCR is a viable technique that can be effectively utilized to obtain unknown DNA sequences. The experimental conditions established in this study serve as a theoretical basis for the amplification of unknown genome sequences of Gramineae crops and other species.