The recent invasion of the hard tick Ixodes affinis, a sylvatic vector of Borrelia burgdorferi, into the Mid-Atlantic region of the United States has prompted questions as to the origin of these ticks, and the possibility of connectivity with presumed ancestral populations in Central and South America. To assess connectivity and ancestry of I. affinis throughout its range, a fragment of the 16S mitochondrial rRNA gene was sequenced from seven adult I. affinis ticks collected in Colon, Panama, and compared to 19 previously published 16S haplotypes in two clades from the US, seven previously published haplotypes from Belize, and one previously published haplotype from Colombia. This study did not find any overlapping haplotypes between the US, Panama, Belize, and Colombia, showing no evidence for connectivity between North, Central, and South American populations of I. affinis. Two new I. affinis clades associated with ticks from Panama and Belize were also identified. The node that gave rise to the Panama clade of I. affinis was more basal than the nodes leading to the clades containing the majority of ticks from Belize and the United States, and ticks from Belize were more closely related to US ticks than Panama ticks. Several possible competing migration pathways were identified, where ticks may have been introduced to the northern US from Belize, or to the southern US from Panama. Star clusters present in each clade suggest rapid mutation rates after arrival in new areas, and may be associated with the success of I. affinis in invading northern climates. This study provides preliminary evidence for the spread of I. affinis from Central America into the US, and serves as a first step in investigating the possibility of connectivity between Central and North America tick populations.