The dynamic characteristics of ship structures are becoming more important as the flexibility of modern ships increases, for example, to predict reliable design life. This requires an accurate dynamic model of the structure, which, because of complex vibration environment and complex boundary conditions, can only be validated by measurements. In the present paper the use of operational modal analysis (OMA) for dynamic characterization of a ship structure based on experimental data, from a full-scale measurement of a 210-m long Ro-Lo ship during sea trial, is presented. The measurements contain three different data sets obtained under different operating conditions of the ship: 10 knots cruising speed, 18 knots cruising speed, and at anchor. Natural frequencies, modal damping ratios, and mode shapes have been successfully estimated for the first 10 global modes. Damping ratios for the current ship were found within the range 0.9%‐1.9% and natural frequencies were found to range from 0.8 to 4.1 Hz for the first 10 global modes of the ship at design speed (18 knots). The three different operating conditions showed, in addition, a speed dependency of the natural frequencies and damping ratios. The natural frequencies were found to be lower for the 18-knots condition compared with the two other conditions, most significantly for the vertical bending modes. Also, for the vertical bending modes, the damping ratios increased by 28%‐288% when the speed increased from 10 to 18 knots. Other modes were not found to have the same strong speed dependency.