The present study describes the frequency of Foot and Mouth Disease (FMD) virus serotypes (O, A and Asia-1) in major regions (all provinces) of Pakistan using Indirect Sandwich ELISA. Also, spatial distribution of various FMD serotypes and their comparison is discussed. A total of 590 samples (Epithelial tissue) have been analyzed during a period of five years (2005–2009). Out of 590 samples, 180 were found positive, giving an overall confirmation of FMDV about 33.2 %. Of the prevalent serotypes, FMDV ‘O’ serotype caused most outbreaks (20.7 %), followed by serotype A (6.6 %) and serotype Asia-1 (4.6 %) while there was no positive case of type ‘C’. The study clearly showed that the disease was more frequent in the agro-climatic zones than in hilly areas. Based on the data of 590 samples (>50 outbreaks), the overall prevalence of FMDV in cattle and buffaloes in Pakistan was 33.2 %, while in cattle alone, it was 37.1 %, higher than in buffalo (28.7 %). There were eight cases of mixed serotypes infection, indicating the presence of endemic state of disease. Another significant feature was the change over time. In phase-I (2005–2007), there was an overall prevalence of 29.4 %, while the occurrence of the serotype O, A and Asia-1 was 20.4 %, 2.9 % and 4.7 %, respectively. During phase-II (2008–2009), the overall prevalence was 59.21 %, while those of serotype O, A and Asia-1 were 22.4 %, 31.6 % and 4.0 %, respectively. This clearly indicated a shift from serotype O to A, which may help to explain the occurrence of more severe outbreaks, despite vaccination.