Various culture‐based methodologies are used in assessment of antibiotic resistance in samples collected in agroecosystems. Culture‐based methods commonly involve isolating target bacteria on general or selective media and assessing growth in response to specific concentrations of antibiotics. The advantages of culture‐based methods are multifold. In particular, isolation of bacteria is key to understanding phenotypic characteristics of isolates and their resistance patterns, and most national and international antibiotic resistance monitoring projects are isolate based. This review covers current knowledge of bacterial groups and antibiotics commonly targeted in resistance studies using bacterial culture and discusses the range in methods used, data interpretation, and factors supporting and confounding the use of culture‐based methods in assessment of antibiotic resistance. Gaps in knowledge related to study design and resistance databases are discussed. Finally, a case is made for the integration of culture‐based and molecular methods to better inform our understanding of antibiotic resistance in agroecosystems.