<p>Recently, the trend analysis of climatic variables has received significant attention due to climate change. Therefore, trend analysis of reference evapotranspiration (ET_o) is pertinent to the sustainable management of water resources on a regional scale. Thus, this study examines the temporal pattern of trend and magnitude in average annual ET_o in five different agro-climatic zones of Punjab (India) by obtaining data from seven locations for a period of 40 years (1981–2020). The ET_o was computed using the Food and Agriculture Organization-56 Penman-Monteith ((FAO-56 (PM)) method with the available climatic data (minimum and maximum temperature, relative humidity, wind speed, and solar radiation). The trend was analyzed using two non-parametric methods namely Mann-Kendall (MK), and Innovative Trend Analysis (ITA) at 5% significance level, while the magnitude (mm/year) of the ET_o trend was estimated using the Sen’s Slope Estimator (SSE) test. The results of the MK and ITA tests revealed a statistically significant negative (downward) trend for all seven locations at 5% significance level. Similarly, the SSE test also indicates the declining magnitude of the trend with a rate of -0.291 to -0.190&#xa0;mm/year across all the locations. In addition, factors influencing the ET_o for the same locations were also analyzed using the stepwise regression technique, and their results demonstrated that the maximum temperature is the most dominating variable with standardized coefficient values of 1.085, 1.066, 1.055, 0.991, 0.978, 0.853, and 0.932 for Ferozepur, Mansa, Amritsar, Ludhiana, Patiala, Rupnagar, and Gurdaspur, respectively. Overall, the findings of this study will help to manage sustainable water resources for agricultural practices by understanding the potential and possible future footprint of climate change on reference evapotranspiration in the study region.</p>