The karyotypes of 5 species of Amblyomma ticks found in North America were examined for the presence and location of constitutive heterochromatin (C-bands). All specimens examined had 20 autosomes and an XX:XO sex-determining system, with the X being the largest element in the karyotype. Except as noted in text, the autosomes of all species were acrocentric and heterochromatin was restricted to the centromeric region. The third largest autosome in 4 of the 5 species displayed a consistent band of heterochromatin just below the centromere; Amblyomma imitator displayed heterochromatin on the third largest pair of autosomes but in the form of an entire heterochromatic short arm. It is hypothesized that the third largest autosome could be the location of the nucleolar organizer region. Unique features of the C-banded karyotype that characterize and differentiate each species are presented. Similarity in the C-banding pattern exists between Amblyomma americanum and Amblyomma maculatum (the C-band in the X chromosome) and Amblyomma cajennense and A. imitator (the extreme subacrocentric X chromosome). The placement of Amblyomma inornatum remains difficult and cannot be clearly determined based on the placement of heterochromatin.