The US Department of Agriculture says the horses are located in at least 27 states.
"Only 12 states have not been involved in the CEM investigation process to date," the department said.
The first case of the venereal disease was confirmed on December 15, in a quarter horse stallion on a central Kentucky property.
There are now seven stallions that have tested positive for CEM - four in Kentucky and three in Indiana.
"The Indiana stallions spent time on the index [original] premises in Kentucky during the 2008 breeding season.
"There are six other exposed stallions and 22 exposed mares currently located in Kentucky."
An exposed horse is one that was on the index premises in Kentucky and/or one that was bred to a CEM-positive horse, either naturally or via artificial insemination.
Outside of Kentucky, the location of 78 exposed horses plus three positive stallions have now been confirmed. The total of 81 horses includes 12 stallions and 69 mares. The 81 horses are located in 27 different states.
"All CEM-positive horses, and all exposed horses that have been located, are currently under quarantine or hold order."
Testing and/or treatment protocols are being put in place for all located horses.
"At least 250 additional horses are actively being traced, with owners of those horses located in at least 27 states," the department says.
CEM is an inflammation of the endometrium of mares caused by Taylorella equigenitalis. It is a highly contagious venereal disease, which usually results in temporary infertility.
Its effects of which are restricted to the reproduction tract of the mare. The disease is resident in mares and transmitted by stallions with infection being spread from the stallion/teaser to mare or from mare to stallion/teaser during mating or teasing.
The bacteria can also be spread via semen collected for artificial insemination.
CEM can be treated with disinfectants and antibiotics.
CEM-positive mares, and mares from CEM-positive countries, are required to go through a treatment protocol and remain in quarantine for no fewer than 21 days.
Stallions that have CEM or come from a CEM-positive country are required to remain quarantined until a treatment protocol is completed and they test negative for the disease.