He studied in Berlin before becoming a researcher and professor at the University of Pennsylvania, where he primarily worked until his death at the age of 39.

His father, Thomas Harrison Montgomery Sr., was a businessman and writer who authored several historical accounts[a] and was president of the Insurance Company of North America from 1882 until his death in 1905.

His most notable research includes early observations of the pairing of maternal and paternal chromosomes during cell division.

[8] He also detailed the morphology of the nucleolus, and observed that in some hemipteran insects the germ cells of males but not females contain odd numbers of chromosomes, which is now known to influence sex-determination,[9][10] A resolution of the American Society of Zoologists read after his death stated "it would be impossible to write a text-book upon the role of the chromosomes in the determination of sex without referring to his crucial labors in this field.

[10] Montgomery wrote 14 scientific articles on spiders, and he was known to keep large amounts in his laboratory and home from which he recorded observations of courtship, mating, and other behaviors.

In a 1909 paper detailing the anatomy and development of various organs in spiders he rejected a prevalent idea at the time that arachnids evolved from Merostomata[b] (a now obsolete group including horseshoe crabs and the extinct eurypterids) adapting to a terrestrial life, and proposed instead that the aquatic lifestyle of horseshoe crabs evolved from terrestrial ancestors.

[17] Montgomery also published on a variety of other topics including principles of animal classification and larval development of the red-backed salamander.

[19] Montgomery was stricken with pneumonia on February 15, 1912, and died in a Philadelphia hospital on March 19, only a few days after his thirty-ninth birthday.