Will moving a hard drive to a different SATA connection on the motherboard affect performance?

Which motherboard and Hard Disk you are using. Please specify the specifications here. Without these there will be problem telling the answer, but remember that your HDD have to support the 6gb/s speed, and if you put the 3gb/s HDD into 6gb/s SATA port, then there will be certainly some speed issues. Always insert the HDD in the same SATA port of same speed. Do reply if u need any help.