The paper, based on RCA, LFI and GL indices, and logistic regression analysis, allowed us to track the dynamics of the comparative advantage/disadvantage of the Czech Republic in individual commodity aggregates in relation to individual EU partners, and to comprehend the role of the main production factors. The only production factor with a statistically significant effect is capital. Trade with countries richly endowed with agricultural land and capital, in most cases, appeared not favorable for the Czech Republic in terms of having a comparative advantage over them, if measured with LFI index. The values of the GL index disclosed that trade with these countries is mostly of an inter-industry character. The analysis revealed the following top-3 mostly competitive Czech aggregates: S3-08 (animal feed stuff), S3-06 (sugar and sugar preparations), along with S3-04 (cereals and cereal preparations). Some aggregates appeared to not have any comparative advantage: S3-01 (meat, meat preparations), S3-05 (vegetables and fruits), and S3-41 (animal oils and fats). Speaking of developmental trends, from 2000 to 2019 the Czech Republic has managed to improve its position in agri-food trade within the EU-27 by 43.81% if measured with LFI, and by 51.63% if measured with RCA. These positive changes also appeared to be statistically significant.