As a raw material for beer, barley seeds play a critical role in producing beers with various flavors. Unexcepted mixed varieties of barley seeds make malt quality uncontrollable and can even destroy beer flavors. To ensure the quality and flavor of malts and beers, beer brewers will strictly check the appropriate varieties of barley seeds during the malting process. There are wide varieties of barley seeds with small sizes and similar features. Professionals can visually distinguish these varieties, which can be tedious and time-consuming and have high misjudgment rates. However, biological testing requires professional equipment, reagents, and laboratories, which are expensive. This study aims to build an automatic artificial intelligence detection method to achieve high performance in multi-barley seed datasets. There are nine varieties of barley seeds (CDC Copeland, AC Metcalfe, Hockett, Scarlett, Expedition, AAC Synergy, Celebration, Legacy, and Tradition). We captured images of these original barley seeds using an iPhone 11 Pro. This study used two mixed datasets, including a single-barley seed dataset and a multi-barley seed dataset, to improve the detection accuracy of multi-barley seeds. The multi-barley seed dataset had random amounts and varieties of barley seeds in each image. The single-barley seed dataset had one barley seed in each image. Data augmentation can reduce overfitting and maximize model performance and accuracy. Multi-variety barley seed recognition deploys an efficient data augmentation method to effectively expand the barley dataset. After adjusting the hyperparameters of the networks and analyzing and augmenting the datasets, the YOLOv5 series network was the most effective in training the two barley seed datasets and achieved the highest performance. The YOLOv5x6 network achieved the second highest performance. The mAP (mean Average Precision) of the trained YOLOv5x6 was 97.5%; precision was 98.4%; recall was 98.1%; the average speed of image detection reached 0.024s. YOLOv5x6 only trained the multi-barley seed dataset; the trained performance was greater than that of the YOLOv5 series. The two datasets had 39.5% higher precision, 27.1% higher recall, and 40.1% higher mAP than when just using the original multi-barley seed dataset. The multi-barley seed detection results showed high performance, robustness, and speed. Therefore, malting and brewing industries can assess the original barley seed quality with the assistance of fast, intelligent, and detected multi-barley seed images.