High resolution melting curve analysis (HRM) is an emerging new method for interrogating and characterizing DNA samples. It has been used as a powerful tool for gene mutation and single-nucleotide polymorphism (SNP) detection with high throughput and low cost. Commercially available HRM analysis systems are mostly proprietary and expensive. It lacks the flexibility for the end users and researchers to incorporate new analysis algorithms into the existing system. This paper presents the development of a MATLAB-based open software program for high resolution melting curve analysis. Key analysis functions, such as to obtain the first derivative curve using Savitzky–Golay filter, to identify the melt region, subtraction of background fluorescence and curve normalization, are introduced, followed by case studies of HRM analysis using the developed software program.