In the present study, investigation of chaotic flow in a two-dimensional closed-loop pulsating heat pipe has been carried out numerically. Constant temperature boundary conditions have been applied for the heating (evaporator) and the cooling (condenser) sections. The width of the tube was 3 mm and water was used as working fluid. Structured meshing configuration has been used and Volume of Fluid (VOF) method has been employed for two-phase flow simulation. The investigated temperature ranges for the evaporator and the condenser were 100–180 °C and 20–50 °C, respectively. The range of filling ratio was from 30 to 80%. Volume fraction of liquid and vapor in the pulsating heat pipe was investigated for different operating conditions. Time series analysis of the adiabatic wall temperature, correlation dimension, power spectrum density, Lyapunov exponent and autocorrelation function were used to investigate chaos in the pulsating heat pipe. Chaotic behavior was observed under several operating conditions. It was found that the time series has complicated, irregular and aperiodic behavior. Absence of dominating peaks in the power spectrum density curves denoted the existence of chaos in the pulsating heat pipe. It was observed that by increasing the filling ratio and evaporator temperature, the correlation dimension increases. Positive values of Lyapunov exponent were obtained for temperatures between 140 and 180 °C at filling ratio of 30%. At filling ratio of 75%, all of the Lyapunov exponents were positive for the temperature interval between 120 and 180 °C.