Endoscopic ultrasonography (EUS) has greater spatial resolution than other diagnostic imaging modalities. In addition, if gallbladder lesions are found and gallbladder cancer is suspected, EUS is an indispensable modality, enabling detailed tests for invasion depth evaluation using the Doppler mode and ultrasound agents. Furthermore, for gallbladder lesions, EUS fine-needle aspiration (EUS-FNA) can be used to differentiate benign and malignant forms of conditions, such as xanthogranulomatous cholecystitis, and collect evidence before chemotherapy. EUS-FNA is also useful for highly precise and specific diagnoses. However, the prevention of bile leakage, an accidental symptom, is highly important. Advancements in next-generation sequencing (NGS) technologies facilitate the application of multiple parallel sequencing to EUS-FNA samples. Several biomarkers are expected to stratify treatment for gallbladder cancer; however, NGS can unveil potential predictive genomic biomarkers for the treatment response. It is believed that NGS may be feasible with samples obtained using EUS-FNA, further increasing the demand for EUS-FNA.