Cotton is an excessive growing fiber crop in the world. In Pakistan, climate warming is hurting the cotton crop. To identify countermeasures of climate change impacts on crop production, it is needed to explore the changes in crop yield and their relationship to climate change. This study, we conducted spatial analysis of cotton yield and climate data in the Indus River basin in Pakistan from 1989- 2018. We analyzed the trend in cotton production indices and climate variables using the Mann-Kendal nonparametric test, and the impact of climate change on cotton yield through Pearson’s correlation and best-subset multivariate linear regression model. Sunshine hours positively impacted on cotton yield, but an increasing trend in average temperature, minimum temperature and air relative humidity in main crop areas had a negative impact on the cotton crop. Our results illustrate the relationship between the cotton yield and climate change variables on regional basis in the Indus River basin, Pakistan. This research could provide a reference for understanding the intolerance of cotton in the Indus River basin, Pakistan to climate change.