All seven pups, who have three sets of biological parents, are said to be healthy and doing well at Cornell University in New York.
They were born after 19 embryos created by in-vitro fertilisation were transferred to the host mother, a beagle.
Two were the result of pairing eggs and sperm from a beagle mother and cocker spaniel father. The other five were from two pairings of beagle mothers and fathers.
Scientists say the experiment opens the door to conserving endangered dog species, eradicating heritable diseases in dogs, and using dogs or their embryos to study human diseases.
Dogs share more than 350 heritable disorders and traits with humans — almost twice as many as any other species.
Lead researcher Dr Alex Travis, from Cornell’s college of veterinary medicine, said: “Since the mid-1970s, people have been trying to do this in a dog and have been unsuccessful.”
The scientists used similar techniques as those offered to childless couples in IVF clinics, with some important differences.
7 Bundles Of Scientific Joy: 'Test Tube Puppies' Prove IVF Can Work In Dogs https://t.co/PVzzUl5sCp— NPR Science Desk (@nprscience) December 9, 2015
Two of the biggest hurdles were ensuring the eggs had reached the right stage of maturation, and simulating the way the female reproductive tract prepared sperm for fertilisation.
“We made those two changes, and now we achieve success in fertilisation rates at 80% to 90%,” said Dr Travis.
Freezing and storing the embyros allowed the scientists to insert them into the surrogate mother’s oviducts at the right time in her reproductive cycle, which occurs only once or twice a year.
Success followed four failed attempts.
IVF will in future allow conservationists to ensure the survival of endangered dog species.
Combined with new gene-editing techniques, it could also help scientists eradicate harmful inherited traits.
In the quest for desired characteristics, inbreeding has burdened some domestic dogs with genetic weakness and disorders.
For instance, golden retrievers are at risk of developing lymphoma while Dalmatians carry a gene that makes them vulnerable to kidney and bladder stones.
“With a combination of gene-editing techniques and IVF, we can potentially prevent genetic disease before it starts,” said Dr Travis.