In mitral stenosis (MS) patients, determining stenosis severity in atrial fibrillation (AF) is time-consuming by averaging measurement by multiple cardiac cycles. Whether a single beat method can be used to measure the mitral valve area (MVA) and transmitral gradient for stenosis severity assessment in AF is still uncertain. Forty-eight MS patients with AF (mean age 46.8+8.6 years) underwent routine echocardiographic study. The MVA by pressure half time (PHT) and transmitral mean gradient were measured by four methods: the standard approach (averaging multiple beats), single short R-R cycle, single long R-R cycle, and single beat RRp/RRpp = 1. 2D and 3D planimetry MVA were measured at the mitral orifice. The single beat RRp/RRpp = 1 showed no significant difference in MVA PHT measurement compared with standard approach (0.8 (0.3 – 2.7 vs 0.9 + 0.3 cm2) cm2, P = 0.472), whereas there was a significant difference in MVA by PHT when short R-R cycle (1.0 (0.4 – 2.7) cm2, P = 0.0001) and long R-R cycle (0.8 (0.3 – 1.7) cm2, P = 0.013) were selected. There was a significant difference in mean MVG measurement when short R-R cycle (12.1 + 3.9 mmHg, P = 0.001), long R-R cycle (10.1 + 4.0 mmHg, P = 0.007), and single beat RRp/RRpp = 1 (12.2 + 4.4 mmHg, P = 0.0001) were selected. Correlation coefficients for MVA PHT calculated by single beat RRp/RRpp = 1 compared with MVA PHT measured by standard approach are r = 0.87 (P < 0.001). There is weak correlation in measurement MVA between PHT single beat RRp/RRpp = 1 and 3D planimetry (r = 0.316, P = 0.044). In AF, the single beat method RRp/RRpp = 1 for measurement MVA by PHT in MS has a high correlation with the current standard approach by averaging multiple beats. Compared with MVA by 3D planimetry as the reference measurement of MVA, measurement of MVA by PHT standard approach and single beat RRp/RRpp = 1 have a weak correlation in AF patients. MVA by planimetry especially 3D planimetry, is considered as a reference measurement, but MVA by PHT single beat RRp/RRpp =1 can be an alternative, especially when MVA by planimetry is not feasible.