An MD5 checksum is a unique 32-character signature (or hash) of a file. It's statistically impossible for two non-identical files to have the same signature. Some download sites publish the MD5 hash of their files, letting you compare the result after downloading to ensure the file you have is identical. You can use this shell extension to do that, or to determine the MD5 hash of files you're uploading.
Two other widely-used hashes are the CRC32, or Cyclic Redundancy Check, and SHA1, and this tool will also calculate the CRC and SHA1 hashes for a file.