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Try right-clicking your definition file, clicking Edit, and either adjusting your destination as needed or simply clicking OK again. If that doesn't fix it, what is the destination of your backup? Local drive or network location?
If it's a local drive, check Other Tasks > Edit Defaults > Advanced > Destination drive discovery. If it's set to the default drive letter, check the drive letter specified in your destination against the drive letter currently assigned to your intended destination. Windows does not always assign the same drive letter to the same device. If you're using volume identifier targeting, have you recently formatted or replaced your destination? If so, editing and re-saving your definition file while that destination is connected and specified (by drive letter) in the Reflect interface will cause the definition file to be updated with that target's unique volume identifier.
If it's a network location, do you have the appropriate credentials for your network location stored under Edit Defaults > Network?