The recently described Lyme disease spirochete Borrelia mayonii is associated with human illness in the Upper Midwest of the United States. Experimental laboratory studies and field observations on natural infection indicate that B. mayonii is maintained by horizontal transmission between tick vectors and vertebrate reservoirs. While maintaining a colony of Ixodes scapularis Say (Acari: Ixodidae) ticks infected with the B. mayonii type strain (MN141420), we had an opportunity to examine whether infected females may pass this spirochete transovarially to their offspring. We found no evidence of B. mayonii infection in subsets of larvae originating from 18 infected I. scapularis females (grand total of 810 larvae tested), or in mice exposed to larval feeding.