Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as Hpa II and Msp I) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both Eco RI/Hpa II and Eco RI/Msp I digested samples. Comparative analysis between Eco RI/Hpa II and Eco RI/Msp I fragment patterns allows the identification of two types of polymorphisms: (1) “Methylation-insensitive polymorphisms” that show common Eco RI/Hpa II and Eco RI/Msp I patterns but are detected as polymorphic amplified fragments among samples; and (2) “Methylation-sensitive polymorphisms” that are associated with amplified fragments differing in their presence or absence or in their intensity between Eco RI/Hpa II and Eco RI/Msp I patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.